Protein kinase stress-related polypeptides and methods of use in plants

ABSTRACT

A transgenic plant transformed by a Protein Kinase Stress-Related Polypeptide (PKSRP) coding nucleic acid, wherein expression of the nucleic acid sequence in the plant results in increased tolerance to environmental stress as compared to a wild type variety of the plant. Also provided are agricultural products, including seeds, produced by the transgenic plants. Also provided are isolated PKSRPs, and isolated nucleic acid coding PKSRPs, and vectors and host cells containing the latter.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/292,408, filed Nov. 12, 2002 and now U.S. Pat. No. 7,176,026, whichclaims the priority benefit of U.S. Provisional Patent Application Ser.No. 60/346,096 filed Nov. 9, 2001. This application is copending withallowed U.S. patent application Ser. No. 11/609,353, filed Dec. 12,2006, which is also a divisional of U.S. Pat. No. 7,176,026. The entirecontents of each priority application identified above are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to nucleic acid sequences encodingpolypeptides that are associated with abiotic stress responses andabiotic stress tolerance in plants. In particular, this inventionrelates to nucleic acid sequences encoding polypeptides that conferdrought, cold, and/or salt tolerance to plants.

2. Background Art

Abiotic environmental stresses, such as drought stress, salinity stress,heat stress, and cold stress, are major limiting factors of plant growthand productivity. Crop losses and crop yield losses of major crops suchas soybean, rice, maize (corn), cotton, and wheat caused by thesestresses represent a significant economic and political factor andcontribute to food shortages in many underdeveloped countries.

Plants are typically exposed during their life cycle to conditions ofreduced environmental water content. Most plants have evolved strategiesto protect themselves against these conditions of desiccation. However,if the severity and duration of the drought conditions are too great,the effects on development, growth, and yield of most crop plants areprofound. Continuous exposure to drought conditions causes majoralterations in the plant metabolism which ultimately lead to cell deathand consequently yield losses.

Developing stress-tolerant plants is a strategy that has the potentialto solve or mediate at least some of these problems. However,traditional plant breeding strategies to develop new lines of plantsthat exhibit resistance (tolerance) to these types of stresses arerelatively slow and require specific resistant lines for crossing withthe desired line. Limited germplasm resources for stress tolerance andincompatibility in crosses between distantly related plant speciesrepresent significant problems encountered in conventional breeding.Additionally, the cellular processes leading to drought, cold, and salttolerance in model drought- and/or salt-tolerant plants are complex innature and involve multiple mechanisms of cellular adaptation andnumerous metabolic pathways. This multi-component nature of stresstolerance has not only made breeding for tolerance largely unsuccessful,but has also limited the ability to genetically engineer stresstolerance plants using biotechnological methods.

Drought and cold stresses, as well as salt stresses, have a common themeimportant for plant growth, and that is water availability. Plants areexposed during their entire life cycle to conditions of reducedenvironmental water content, and most plants have evolved strategies toprotect themselves against these conditions of desiccation. However, ifthe severity and duration of the drought conditions are too great, theeffects on plant development, growth and yield of most crop plants areprofound. Furthermore, most of the crop plants are very susceptible tohigher salt concentrations in the soil. Because high salt content insome soils results in less water being available for cell intake, highsalt concentration has an effect on plants similar to the effect ofdrought on plants. Additionally, under freezing temperatures, plantcells lose water as a result of ice formation that starts in theapoplast and withdraws water from the symplast. A plant's molecularresponse mechanisms to each of these stress conditions are common, andprotein kinases play an essential role in these molecular mechanisms.

Protein kinases represent a superfamily, and the members of thissuperfamily catalyze the reversible transfer of a phosphate group of ATPto serine, threonine, and tyrosine amino acid side chains on targetpolypeptides. Protein kinases are primary elements in signalingprocesses in plants and have been reported to play crucial roles inperception and transduction of signals that allow a cell (and the plant)to respond to environmental stimuli. In particular, receptor proteinkinases (RPKs) represent one group of protein kinases that activate acomplex array of intracellular signaling pathways in response to theextracellular environment (Van der Gear et al., 1994, Annu. Rev. CellBiol. 10:251-337). RPKs are single-pass transmembrane polypeptides thatcontain an amino-terminal signal sequence, extracellular domains uniqueto each receptor, and a cytoplasmic kinase domain. Ligand bindinginduces homo- or hetero-dimerization of RPKs, and the resultant closeproximity of the cytoplasmic domains results in kinase activation bytransphosphorylation. Although plants have many polypeptides similar toRPKs, no ligand has been identified for these receptor-like kinases(RLKs). The majority of plant RLKs that have been identified belong tothe family of Serine/Threonine (Ser/Thr) kinases, and most haveextracellular Leucine-rich repeats (Becraft, P W., 1998, Trends PlantSci. 3:384-388).

Another type of protein kinase is the Ca+-dependent protein kinase(CDPK). This type of kinase has a calmodulin-like domain at the COOHterminus which allows response to Ca+ signals directly withoutcalmodulin being present. Currently, CDPKs are the most prevalentSer/Thr polypeptide kinases found in higher plants. Although theirphysiological roles remain unclear, they are induced by cold, drought,and abscisic acid (ABA) (Knight et al., 1991, Nature 352:524; Schroeder,J. J. and Thuleau, P., 1991, Plant Cell 3:555; Bush, D. S., 1995, Annu.Rev. Plant Phys. Plant Mol. Biol. 46:95; Urao, T. et al., 1994, Mol.Gen. Genet. 244:331).

Another type of signaling mechanism involves members of the conservedSNF1 Serine/Threonine polypeptide kinase family. These kinases playessential roles in eukaryotic glucose and stress signaling. PlantSNF1-like kinases participate in the control of key metabolic enzymes,including HMGR, nitrate reductase, sucrose synthase, and sucrosephosphate synthase (SPS). Genetic and biochemical data indicate thatsugar-dependent regulation of SNF1 kinases involves several othersensory and signaling components in yeast, plants, and animals.

Additionally, members of the Mitogen-Activated Protein Kinase (MAPK)family have been implicated in the actions of numerous environmentalstresses in animals, yeasts and plants. It has been demonstrated thatboth MAPK-like kinase activity and mRNA levels of the components of MAPKcascades increase in response to environmental stress and plant hormonesignal transduction. MAP kinases are components of sequential kinasecascades, which are activated by phosphorylation of threonine andtyrosine residues by intermediate upstream MAP kinase kinases (MAPKKs).The MAPKKs are themselves activated by phosphorylation of serine andthreonine residues by upstream kinases (MAPKKKs). A number of MAP Kinasegenes have been reported in higher plants.

Another major type of environmental stress is lodging, which refers tothe bending of shoots or stems in response to wind, rain, pests ordisease. Two types of lodging occur in cereals: root-lodging and stembreakage. The most common type of lodging is root lodging, which occursearly in the season. Stem-breakage, by comparison, occurs later in theseason as the stalk becomes more brittle due to crop maturation. Stembreakage has greater adverse consequences on crop yield, since theplants cannot recover as well as from the earlier root-lodging.

Lodging in cereal crops is influenced by morphological (structural)plant traits as well as environmental conditions. Lodging in cereals isoften a result of the combined effects of inadequate standing power ofthe crop and adverse weather conditions, such as rain, wind, and/orhail. Lodging is also variety (cultivar) dependent. For example, a tall,weak-stemmed wheat cultivar has a greater tendency to lodge than asemi-dwarf cultivar with stiffer straw. In addition, the tendency of acrop to lodge depends on the resistance especially of the lowerinternodes. This is because the lower internodes have to resist thegreatest movement of force. The weight of the higher internodes of thestems plus leaves and heads in relation to the stem (culm) will affectthe resistance of a crop to lodging. The heavier the higher parts of thestem are and the greater the distance from their center of gravity tothe base of the stem, the greater is the movement of the forces actingupon the lower internodes and the roots. Supporting this argument, itwas found that the breaking strength of the lowest internode and shootper root ratio were the most suitable indices of lodging. Furthermore,plant morphological (structural) characteristics such as plant height,wall thickness, and cell wall lignification can affect the ability ofthe plant to resist a lateral force.

Severe lodging is very costly due to its effects on grain formation andassociated harvesting problems and losses. It takes about twice the timeto harvest a lodged crop than a standing one. Secondary growth incombination with a flattened crop makes harvesting difficult and cansubsequently lead to poor grain quality. Yield loss comes from poorgrain filling, head loss, and bird damage. Yield losses are most severewhen a crop lodges during the ten days following head emergence. Yieldlosses at this stage will range between 15% and 40%. Lodging that occursafter the plant matures will not affect the yield but it may reduce theamount of harvestable grain. For instance, when lodging occurs after theplant matures, neck breakage and the loss of the whole head can result;these often lead to severe harvest losses. In theses cases, farmers whostraight combine their grain will likely incur higher losses than thosewho swath them. Accordingly, it is desirable to identify genes expressedin lodging resistant plants that have the capacity to confer lodgingresistance to the host plant and to other plant species.

Although some genes that are involved in stress responses in plants havebeen characterized, the characterization and cloning of plant genes thatconfer stress tolerance remains largely incomplete and fragmented. Forexample, certain studies have indicated that drought and salt stress insome plants may be due to additive gene effects, in contrast to otherresearch that indicates specific genes are transcriptionally activatedin vegetative tissue of plants under osmotic stress conditions. Althoughit is generally assumed that stress-induced proteins have a role intolerance, direct evidence is still lacking, and the functions of manystress-responsive genes are unknown.

There is a need, therefore, to identify genes expressed in stresstolerant plants that have the capacity to confer stress tolerance to itshost plant and to other plant species. Newly generated stress tolerantplants will have many advantages, such as an increased range in whichthe crop plants can be cultivated, by for example, decreasing the waterrequirements of a plant species. Other desirable advantages includeincreased resistance to lodging, the bending of shoots or stems inresponse to wind, rain, pests, or disease.

SUMMARY OF THE INVENTION

This invention fulfills in part the need to identify new, unique proteinkinases capable of conferring stress tolerance to plants uponover-expression. The present invention describes a novel genus ofProtein Kinase Stress-Related Polypeptides (PKSRPs) and PKSRP codingnucleic acids that are important for modulating a plant's response to anenvironmental stress. More particularly, over-expression of these PKSRPcoding nucleic acids in a plant results in the plant's increasedtolerance to an environmental stress.

The present invention includes an isolated plant cell comprising a PKSRPcoding nucleic acid, wherein expression of the nucleic acid sequence inthe plant cell results in increased tolerance to environmental stress ascompared to a wild type variety of the plant cell. Namely, describedherein are PK-3, PK-4, PK-10, and PK-11 from Physcomitrella patens;BnPK-1, BnPK-2, BnPK-3, and BnPK-4 from Brassica napus; GmPK-1, GmPK-2,GmPK-3, and GmPK-4, from Glycine max; and OsPK-1 from Oryza sativa.

The invention provides in some embodiments that the PKSRP and codingnucleic acid are those that are found in members of the genusPhyscomitrella Brassica, Glycine, or Oryza. In another preferredembodiment, the nucleic acid and polypeptide are from a Physcomitrellapatens plant, a Brassica napus plant, a Glycine max plant, or an Oryzasativa plant. The invention provides that the environmental stress canbe increased salinity, drought, temperature, metal, chemical,pathogenic, and oxidative stresses, or combinations thereof. Inpreferred embodiments, the environmental stress can be drought or coldtemperature.

The invention further provides a seed produced by a transgenic planttransformed by a PKSRP coding nucleic acid, wherein the plant is truebreeding for increased tolerance to environmental stress as compared toa wild type variety of the plant. The invention further provides a seedproduced by a transgenic plant expressing a PKSRP, wherein the plant istrue breeding for increased tolerance to environmental stress ascompared to a wild type variety of the plant.

The invention further provides an agricultural product produced by anyof the below-described transgenic plants, plant parts or seeds. Theinvention further provides an isolated PKSRP as described below. Theinvention further provides an isolated PKSRP coding nucleic acid,wherein the PKSRP coding nucleic acid codes for a PKSRP as describedbelow.

The invention further provides an isolated recombinant expression vectorcomprising a PKSRP coding nucleic acid as described below, whereinexpression of the vector in a host cell results in increased toleranceto environmental stress as compared to a wild type variety of the hostcell. The invention further provides a host cell containing the vectorand a plant containing the host cell.

The invention further provides a method of producing a transgenic plantwith a PKSRP coding nucleic acid, wherein expression of the nucleic acidin the plant results in increased tolerance to environmental stress ascompared to a wild type variety of the plant comprising: (a)transforming a plant cell with an expression vector comprising a PKSRPcoding nucleic acid, and (b) generating from the plant cell a transgenicplant with an increased tolerance to environmental stress as compared toa wild type variety of the plant. In preferred embodiments, the PKSRPand PKSRP coding nucleic acid are as described below.

The present invention further provides a method of identifying a novelPKSRP, comprising (a) raising a specific antibody response to a PKSRP,or fragment thereof, as described below; (b) screening putative PKSRPmaterial with the antibody, wherein specific binding of the antibody tothe material indicates the presence of a potentially novel PKSRP; and(c) identifying from the bound material a novel PKSRP in comparison toknown PKSRP. Alternatively, hybridization with nucleic acid probes asdescribed below can be used to identify novel PKSRP nucleic acids.

The present invention also provides methods of modifying stresstolerance of a plant comprising, modifying the expression of a PKSRPnucleic acid in the plant, wherein the PKSRP is as described below. Theinvention provides that this method can be performed such that thestress tolerance is either increased or decreased. Preferably, stresstolerance is increased in a plant via increasing expression of a PKSRPnucleic acid.

In another aspect, the invention provides methods of increasing aplant's resistance to lodging comprising, transforming a plant cell withan expression cassette comprising a PKSRP nucleic acid and generating aplant from the plant cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagram of the plant expression vector pBPS-JH001containing the super promoter driving the expression of the PKSRP codingnucleic acid (“Gene of Interest”). The components are: aacCI gentamycinresistance gene (Hajdukiewicz et al., 1994, Plant Molec. Biol. 25:989-94), NOS promoter (Becker et al., 1992, Plant Molec. Biol. 20:1195-97), g7T terminator (Becker et al., 1992), and NOSpA terminator(Jefferson et al., 1987, EMBO J. 6:3901-7).

FIG. 2 shows a diagram of the plant expression vector pBPS-SC022containing the super promoter driving the expression of the PKSRP codingnucleic acid (“Gene of Interest”). The components are: NPTII kanamycinresistance gene (Hajdukiewicz et al., 1994, Plant Molec. Biol. 25:989-98), AtAct2-1 promoter (An et al., 1996, Plant J. 10: 107-21), andOCS3 terminator (Weigel et al., 2000, Plant Physiol 122:1003-13).

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of the preferred embodiments of theinvention and the Examples included herein. However, before the presentcompounds, compositions, and methods are disclosed and described, it isto be understood that this invention is not limited to specific nucleicacids, specific polypeptides, specific cell types, specific host cells,specific conditions, or specific methods, etc., as such may, of course,vary, and the numerous modifications and variations therein will beapparent to those skilled in the art. It is also to be understood thatthe terminology used herein is for the purpose of describing specificembodiments only and is not intended to be limiting. In particular, thedesignation of the amino acid sequences as “Protein KinaseStress-Related Polypeptides” (PKSRPs), in no way limits thefunctionality of those sequences.

The present invention describes a novel genus of Protein KinaseStress-Related Polypeptides (PKSRPs) and PKSRP coding nucleic acids thatare important for modulating a plant's response to an environmentalstress. More particularly, over-expression of these PKSRP coding nucleicacids in a plant results in the plant's increased tolerance to anenvironmental stress.

The present invention provides a transgenic plant cell transformed by aPKSRP coding nucleic acid, wherein expression of the nucleic acidsequence in the plant cell results in increased tolerance toenvironmental stress or increased resistance to lodging as compared to awild type variety of the plant cell. The invention further providestransgenic plant parts and transgenic plants containing the plant cellsdescribed herein. In preferred embodiments, the transgenic plants andplant parts have increased tolerance to environmental stress orincreased resistance to lodging as compared to a wild type variety ofthe plant. Plant parts include, but are not limited to, stems, roots,ovules, stamens, leaves, embryos, meristematic regions, callus tissue,gametophytes, sporophytes, pollen, microspores, and the like. In oneembodiment, the transgenic plant is male sterile. Also provided is aplant seed produced by a transgenic plant transformed by a PKSRP codingnucleic acid, wherein the seed contains the PKSRP coding nucleic acid,and wherein the plant is true breeding for increased tolerance toenvironmental stress as compared to a wild type variety of the plant.The invention further provides a seed produced by a transgenic plantexpressing a PKSRP, wherein the seed contains the PKSRP, and wherein theplant is true breeding for increased tolerance to environmental stressas compared to a wild type variety of the plant. The invention alsoprovides an agricultural product produced by any of the below-describedtransgenic plants, plant parts, and plant seeds. Agricultural productsinclude, but are not limited to, plant extracts, proteins, amino acids,carbohydrates, fats, oils, polymers, vitamins, and the like.

As used herein, the term “variety” refers to a group of plants within aspecies that share constant characters that separate them from thetypical form and from other possible varieties within that species.While possessing at least one distinctive trait, a variety is alsocharacterized by some variation between individuals within the variety,based primarily on the Mendelian segregation of traits among the progenyof succeeding generations. A variety is considered “true breeding” for aparticular trait if it is genetically homozygous for that trait to theextent that, when the true-breeding variety is self-pollinated, asignificant amount of independent segregation of the trait among theprogeny is not observed. In the present invention, the trait arises fromthe transgenic expression of one or more DNA sequences introduced into aplant variety.

The present invention describes for the first time that thePhyscomitrella patens PKSRPs, PK-3, PK-4, PK-10, and PK-11; the Brassicanapus PKSRPs, BnPK-1, BnPK-2, BnPK-3, and BnPK-4; the Glycine maxPKSRPs, GmPK-1, GmPK-2, GmPK-3, and GmPK-4; and the Oryza sativa PKSRPOsPK-1 are useful for increasing a plant's tolerance to environmentalstress. As used herein, the term polypeptide refers to a chain of atleast four amino acids joined by peptide bonds. The chain may be linear,branched, circular or combinations thereof. Accordingly, the presentinvention provides isolated PKSRPs selected from the group consisting ofPK-3, PK-4, PK-10, PK-11, BnPK-1, BnPK-2, BnPK-3, BnPK-4, GmPK-1,GmPK-2, GmPK-3, GmPK-4, and OsPK-1, and homologs thereof. In preferredembodiments, the PKSRP is selected from: 1) Physcomitrella patensProtein Kinase-3 (PK-3) polypeptide as defined in SEQ ID NO:3; 2)Physcomitrella patens Protein Kinase-4 (PK-4) polypeptide as defined inSEQ ID NO:6; 3) Physcomitrella patens Protein Kinase-10 (PK-10)polypeptide as defined in SEQ ID NO:9; 4) Physcomitrella patens ProteinKinase-11 (PK-11) polypeptide as defined in SEQ ID NO:12; 5) Brassicanapus Protein Kinase-1 (BnPK-1) polypeptide as defined in SEQ ID NO:14;6) Brassica napus Protein Kinase-2 (BnPK-2) polypeptide as defined inSEQ ID NO:16; 7) Brassica napus Protein Kinase-3 (BnPK-3) polypeptide asdefined in SEQ ID NO:18; 8) Brassica napus Protein Kinase-4 (BnPK-4)polypeptide as defined in SEQ ID NO:20; 9) Glycine max Protein Kinase-1(GmPK-1) polypeptide as defined in SEQ ID NO:22; 10) Glycine max ProteinKinase-2 (GmPK-2) polypeptide as defined in SEQ ID NO:24; 11) Glycinemax Protein Kinase-3 (GmPK-3) polypeptide as defined in SEQ ID NO:26;12) Glycine max Protein Kinase-4 (GmPK-4) polypeptide as defined in SEQID NO:28; 13) Oryza sativa Protein Kinase-1 (OsPK-1) polypeptide asdefined in SEQ ID NO:30; and homologs and orthologs thereof. Homologsand orthologs of the amino acid sequences are defined below.

The PKSRPs of the present invention are preferably produced byrecombinant DNA techniques. For example, a nucleic acid moleculeencoding the polypeptide is cloned into an expression vector (asdescribed below), the expression vector is introduced into a host cell(as described below) and the PKSRP is expressed in the host cell. ThePKSRP can then be isolated from the cells by an appropriate purificationscheme using standard polypeptide purification techniques. For thepurposes of the invention, the term “recombinant polynucleotide” refersto a polynucleotide that has been altered, rearranged or modified bygenetic engineering. Examples include any cloned polynucleotide, andpolynucleotides that are linked or joined to heterologous sequences. Theterm “recombinant” does not refer to alterations to polynucleotides thatresult from naturally occurring events, such as spontaneous mutations.Alternative to recombinant expression, a PKSRP, or peptide can besynthesized chemically using standard peptide synthesis techniques.Moreover, native PKSRP can be isolated from cells (e.g., Physcomitrellapatens, Brassica napus, Glycine max, or Oryza sativa), for example usingan anti-PKSRP antibody, which can be produced by standard techniquesutilizing a PKSRP or fragment thereof.

The invention further provides an isolated PKSRP coding nucleic acid.The present invention includes PKSRP coding nucleic acids that encodePKSRPs as described herein. In preferred embodiments, the PKSRP codingnucleic acid is selected from: 1) Physcomitrella patens Protein Kinase-3(PK-3) nucleic acid as defined in SEQ ID NO:2; 2) Physcomitrella patensProtein Kinase-4 (PK-4) nucleic acid as defined in SEQ ID NO:5; 3)Physcomitrella patens Protein Kinase-10 (PK-10) nucleic acid as definedin SEQ ID NO:8; 4) Physcomitrella patens Protein Kinase-11 (PK-11)nucleic acid as defined in SEQ ID NO:11; 5) Brassica napus ProteinKinase-1 (BnPK-1) nucleic acid as defined in SEQ ID NO:13; 6) Brassicanapus Protein Kinase-2 (BnPK-2) nucleic acid as defined in SEQ ID NO:15;7) Brassica napus Protein Kinase-3 (BnPK-3) nucleic acid as defined inSEQ ID NO:17; 8) Brassica napus Protein Kinase-4 (BnPK-4) nucleic acidas defined in SEQ ID NO:19; 9) Glycine max Protein Kinase-1 (GmPK-1)nucleic acid as defined in SEQ ID NO:21; 10) Glycine max ProteinKinase-2 (GmPK-2) nucleic acid as defined in SEQ ID NO:23; 11) Glycinemax Protein Kinase-3 (GmPK-3) nucleic acid as defined in SEQ ID NO:25;12) Glycine max Protein Kinase-4 (GmPK-4) nucleic acid as defined in SEQID NO:27; 13) Oryza sativa Protein Kinase-1 (OsPK-1) nucleic acid asdefined in SEQ ID NO:29; and homologs and orthologs thereof. Homologsand orthologs of the nucleotide sequences are defined below. In onepreferred embodiment, the nucleic acid and polypeptide are isolated fromthe plant genus Physcomitrella, Brassica, Glycine, or Oryza. In anotherpreferred embodiment, the nucleic acid and polypeptide are from aPhyscomitrella patens (P. patens) plant, a Brassica napus plant, aGlycine max plant, or an Oryza sativa plant.

As used herein, the term “environmental stress” refers to anysub-optimal growing condition and includes, but is not limited to,sub-optimal conditions associated with salinity, drought, temperature,metal, chemical, pathogenic, and oxidative stresses, or combinationsthereof. In preferred embodiments, the environmental stress can beselected from one or more of the group consisting of salinity, drought,or temperature, or combinations thereof, and in particular, can beselected from one or more of the group consisting of high salinity, lowwater content, or low temperature. Also included within the definitionof “environmental stress” is lodging, or the bending of shoots or stemsin response to elements such as wind, rain, pests, or disease.Accordingly, the present invention provides compositions and methods ofincreasing lodging resistance in a plant. It is also to be understoodthat as used in the specification and in the claims, “a” or “an” canmean one or more, depending upon the context in which it is used. Thus,for example, reference to “a cell” can mean that at least one cell canbe utilized.

As also used herein, the term “nucleic acid” and “polynucleotide” referto RNA or DNA that is linear or branched, single or double stranded, ora hybrid thereof. The term also encompasses RNA/DNA hybrids. These termsalso encompass untranslated sequence located at both the 3′ and 5′ endsof the coding region of the gene: at least about 1000 nucleotides ofsequence upstream from the 5′ end of the coding region and at leastabout 200 nucleotides of sequence downstream from the 3′ end of thecoding region of the gene. Less common bases, such as inosine,5-methylcytosine, 6-methyladenine, hypoxanthine and others can also beused for antisense, dsRNA and ribozyme pairing. For example,polynucleotides that contain C-5 propyne analogues of uridine andcytidine have been shown to bind RNA with high affinity and to be potentantisense inhibitors of gene expression. Other modifications, such asmodification to the phosphodiester backlbone, or the 2′-hydroxy in theribose sugar group of the RNA can also be made. The antisensepolynucleotides and ribozymes can consist entirely of ribonucleotides,or can contain mixed ribonucleotides and deoxyribonucleotides. Thepolynucleotides of the invention may be produced by any means, includinggenomic preparations, cDNA preparations, in vitro synthesis, RT-PCR, andin vitro or in vivo transcription.

An “isolated” nucleic acid molecule is one that is substantiallyseparated from other nucleic acid molecules which are present in thenatural source of the nucleic acid (i.e., sequences encoding otherpolypeptides). Preferably, an “isolated” nucleic acid is free of some ofthe sequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in its naturallyoccurring replicon. For example, a cloned nucleic acid is consideredisolated. In various embodiments, the isolated PKSRP nucleic acidmolecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5kb, or 0.1 kb of nucleotide sequences which naturally flank the nucleicacid molecule in genomic DNA of the cell from which the nucleic acid isderived (e.g., a Physcomitrella patens, a Brassica napus, a Glycine max,or an Oryza sativa cell). A nucleic acid is also considered isolated ifit has been altered by human intervention, or placed in a locus orlocation that is not its natural site, or if it is introduced into acell by agroinfection. Moreover, an “isolated” nucleic acid molecule,such as a cDNA molecule, can be free from some of the other cellularmaterial with which it is naturally associated, or culture medium whenproduced by recombinant techniques, or chemical precursors or otherchemicals when chemically synthesized.

Specifically excluded from the definition of “isolated nucleic acids”are: naturally-occurring chromosomes (such as chromosome spreads),artificial chromosome libraries, genomic libraries, and cDNA librariesthat exist either as an in vitro nucleic acid preparation or as atransfected/transformed host cell preparation, wherein the host cellsare either an in vitro heterogeneous preparation or plated as aheterogeneous population of single colonies. Also specifically excludedare the above libraries wherein a specified nucleic acid makes up lessthan 5% of the number of nucleic acid inserts in the vector molecules.Further specifically excluded are whole cell genomic DNA or whole cellRNA preparations (including whole cell preparations that aremechanically sheared or enzymatically digested). Even furtherspecifically excluded are the whole cell preparations found as either anin vitro preparation or as a heterogeneous mixture separated byelectrophoresis wherein the nucleic acid of the invention has notfurther been separated from the heterologous nucleic acids in theelectrophoresis medium (e.g., further separating by excising a singleband from a heterogeneous band population in an agarose gel or nylonblot).

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having a nucleotide sequence of SEQ ID NO:2, SEQ ID NO:5 SEQ IDNO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, or a portion thereof, can be isolated using standard molecularbiology techniques and the sequence information provided herein. Forexample, a P. patens PKSRP cDNA can be isolated from a P. patens libraryusing all or portion of one of the sequences of SEQ ID NO:1 and SEQ IDNO:4. Moreover, a nucleic acid molecule encompassing all or a portion ofone of the sequences of SEQ ID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:29 can beisolated by the polymerase chain reaction using oligonucleotide primersdesigned based upon this sequence. For example, mRNA can be isolatedfrom plant cells (e.g., by the guanidinium-thiocyanate extractionprocedure of Chirgwin et al., 1979, Biochemistry 18:5294-5299) and cDNAcan be prepared using reverse transcriptase (e.g., Moloney MLV reversetranscriptase, available from Gibco/BRL, Bethesda, Md.; or AMV reversetranscriptase, available from Seikagaku America, Inc., St. Petersburg,Fla.). Synthetic oligonucleotide primers for polymerase chain reactionamplification can be designed based upon one of the nucleotide sequencesshown in SEQ ID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:23, SEQ ID NO:25, SEQ ID NO:27, and SEQ ID NO:29. A nucleic acidmolecule of the invention can be amplified using cDNA or, alternatively,genomic DNA, as a template and appropriate oligonucleotide primersaccording to standard PCR amplification techniques. The nucleic acidmolecule so amplified can be cloned into an appropriate vector andcharacterized by DNA sequence analysis. Furthermore, oligonucleotidescorresponding to a PKSRP nucleotide sequence can be prepared by standardsynthetic techniques, e.g., using an automated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises one of the nucleotide sequences shown in SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO: 10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17,SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27,and SEQ ID NO:29. These cDNAs may comprise sequences encoding thePKSRPs, (i.e., the “coding regions” of PK-3 and PK-4), as well as 5′untranslated sequences and 3′ untranslated sequences. The coding regionof PK-3 comprises nucleotides 138-1409 of SEQ ID NO:2 whereas the codingregion of PK-4 comprises nucleotides 142-1395 of SEQ ID NO:5. It is tobe understood that SEQ ID NO:2 and SEQ ID NO:5 comprise both codingregions and 5′ and 3′ untranslated regions. Alternatively, the nucleicacid molecules of the present invention can comprise only the codingregion of any of the sequences in SEQ ID NO:2 and SEQ ID NO:5 or cancontain whole genomic fragments isolated from genomic DNA. The presentinvention also includes PKSRP coding nucleic acids that encode PKSRPs asdescribed herein. Preferred is a PKSRP coding nucleic acid that encodesa PKSRP selected from the group consisting of PK-3 as defined in SEQ IDNO:3, PK-4 as defined in SEQ ID NO:6, PK-10 as defined in SEQ ID NO:9,PK-11 as defined in SEQ ID NO:12, BnPK-1 as defined in SEQ ID NO:14,BnPK-2 as defined in SEQ ID NO:16, BnPK-3 as defined in SEQ ID NO:18,BnPK-4 as defined in SEQ ID NO:20, GmPK-1 as defined in SEQ ID NO:22,GmPK-2 as defined in SEQ ID NO:24, GmPK-3 as defined in SEQ ID NO:26,GmPK-4 as defined in SEQ ID NO:28, and OsPK-1 as defined in SEQ IDNO:30.

Moreover, the nucleic acid molecule of the invention can comprise aportion of the coding region of one of the sequences in SEQ ID NO:2, SEQID NO:5 SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, or SEQ ID NO:29, for example, a fragment which can be used as aprobe or primer or a fragment encoding a biologically active portion ofa PKSRP. The nucleotide sequences determined from the cloning of thePKSRP genes from Physcomitrella patens, Brassica napus, Glycine max, andOryza sativa allow for the generation of probes and primers designed foruse in identifying and/or cloning PKSRP homologs in other cell types andorganisms, as well as PKSRP homologs from other related species. Theportion of the coding region can also encode a biologically activefragment of a PKSRP.

As used herein, the term “biologically active portion of” a PKSRP isintended to include a portion, e.g., a domain/motif, of a PKSRP thatparticipates in modulation of stress tolerance in a plant, and morepreferably, drought tolerance or salt tolerance. For the purposes of thepresent invention, modulation of stress tolerance refers to at least a10% increase or decrease in the stress tolerance of a transgenic plantcomprising a PKSRP expression cassette (or expression vector) ascompared to the stress tolerance of a non-transgenic control plant.Methods for quantitating stress tolerance are provided at least inExample 7 below. In a preferred embodiment, the biologically activeportion of a PKSRP increases a plant's tolerance to an environmentalstress.

Biologically active portions of a PKSRP include peptides comprisingamino acid sequences derived from the amino acid sequence of a PKSRP,e.g., an amino acid sequence of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9,SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20,SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30or the amino acid sequence of a polypeptide identical to a PKSRP, whichinclude fewer amino acids than a full length PKSRP or the full lengthpolypeptide which is identical to a PKSRP, and exhibit at least oneactivity of a PKSRP. Typically, biologically active portions (e.g.,peptides which are, for example, 5, 10, 15, 20, 30, 35, 36, 37, 38, 39,40, 50, 100 or more amino acids in length) comprise a domain or motifwith at least one activity of a PKSRP. Moreover, other biologicallyactive portions in which other regions of the polypeptide are deleted,can be prepared by recombinant techniques and evaluated for one or moreof the activities described herein. Preferably, the biologically activeportions of a PKSRP include one or more selected domains/motifs orportions thereof having biological activity such as a kinase domain. Forexample, the kinase domain of PK-3 spans amino acid residues 87-360 ofSEQ ID NO:3, and the kinase domain of PK-4 spans amino acid residues81-281 of SEQ ID NO:6. Accordingly, the present invention includesPKSRPs comprising amino acid residues 87-360 of SEQ ID NO:3 and aminoacid residues 81-281 of SEQ ID NO:6.

The invention also provides PKSRP chimeric or fusion polypeptides. Asused herein, a PKSRP “chimeric polypeptide” or “fusion polypeptide”comprises a PKSRP operatively linked to a non-PKSRP. A PKSRP refers to apolypeptide having an amino acid sequence corresponding to a PKSRP,whereas a non-PKSRP refers to a polypeptide having an amino acidsequence corresponding to a polypeptide which is not substantiallyidentical to the PKSRP, e.g., a polypeptide that is different from thePKSRP and is derived from the same or a different organism. As usedherein with respect to the fusion polypeptide, the term “operativelylinked” is intended to indicate that the PKSRP and the non-PKSRP arefused to each other so that both sequences fulfill the proposed functionattributed to the sequence used. The non-PKSRP can be fused to theN-terminus or C-terminus of the PKSRP. For example, in one embodiment,the fusion polypeptide is a GST-PKSRP fusion polypeptide in which thePKSRP sequences are fused to the C-terminus of the GST sequences. Suchfusion polypeptides can facilitate the purification of recombinantPKSRPs. In another embodiment, the fusion polypeptide is a PKSRPcontaining a heterologous signal sequence at its N-terminus. In certainhost cells (e.g., mammalian host cells), expression and/or secretion ofa PKSRP can be increased through use of a heterologous signal sequence.

Preferably, a PKSRP chimeric or fusion polypeptide of the invention isproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and re-amplified togenerate a chimeric gene sequence (See, e.g., Current Protocols inMolecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). A PKSRPencoding nucleic acid can be cloned into such an expression vector suchthat the fusion moiety is linked in-frame to the PKSRP.

In addition to fragments and fusion polypeptides of the PKSRPs describedherein, the present invention includes homologs and analogs of naturallyoccurring PKSRPs and PKSRP encoding nucleic acids in a plant. “Homologs”are defined herein as two nucleic acids or polypeptides that havesimilar, or substantially identical, nucleotide or amino acid sequences,respectively. Homologs include allelic variants, orthologs, paralogs,agonists and antagonists of PKSRPs as defined hereafter. The term“homolog” further encompasses nucleic acid molecules that differ fromone of the nucleotide sequences shown in SEQ ID NO:2, SEQ ID NO:5 SEQ IDNO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQ IDNO:29 (and portions thereof) due to degeneracy of the genetic code andthus encode the same PKSRP as that encoded by the nucleotide sequencesshown in SEQ ID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29. As used herein a“naturally occurring” PKSRP refers to a PKSRP amino acid sequence thatoccurs in nature. Preferably, a naturally occurring PKSRP comprises anamino acid sequence selected from the group consisting of SEQ ID NO:3,SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, and SEQ ID NO:30.

An agonist of the PKSRP can retain substantially the same, or a subset,of the biological activities of the PKSRP. An antagonist of the PKSRPcan inhibit one or more of the activities of the naturally occurringform of the PKSRP. For example, the PKSRP antagonist can competitivelybind to a downstream or upstream member of the cell membrane componentmetabolic cascade that includes the PKSRP, or bind to a PKSRP thatmediates transport of compounds across such membranes, therebypreventing translocation from taking place.

Nucleic acid molecules corresponding to natural allelic variants andanalogs, orthologs and paralogs of a PKSRP cDNA can be isolated based ontheir identity to the Physcomitrella patens, Brassica napus, Glycinemax, or Oryza sativa PKSRP nucleic acids described herein using PKSRPcDNAs, or a portion thereof, as a hybridization probe according tostandard hybridization techniques under stringent hybridizationconditions. In an alternative embodiment, homologs of the PKSRP can beidentified by screening combinatorial libraries of mutants, e.g.,truncation mutants, of the PKSRP for PKSRP agonist or antagonistactivity. In one embodiment, a variegated library of PKSRP variants isgenerated by combinatorial mutagenesis at the nucleic acid level and isencoded by a variegated gene library. A variegated library of PKSRPvariants can be produced by, for example, enzymatically ligating amixture of synthetic oligonucleotides into gene sequences such that adegenerate set of potential PKSRP sequences is expressible as individualpolypeptides, or alternatively, as a set of larger fusion polypeptides(e.g., for phage display) containing the set of PKSRP sequences therein.There are a variety of methods that can be used to produce libraries ofpotential PKSRP homologs from a degenerate oligonucleotide sequence.Chemical synthesis of a degenerate gene sequence can be performed in anautomatic DNA synthesizer, and the synthetic gene is then ligated intoan appropriate expression vector. Use of a degenerate set of genesallows for the provision, in one mixture, of all of the sequencesencoding the desired set of potential PKSRP sequences. Methods forsynthesizing degenerate oligonucleotides are known in the art. See,e.g., Narang, S. A., 1983, Tetrahedron 39:3; Itakura et al., 1984, Annu.Rev. Biochem. 53:323; Itakura et al., 1984, Science 198:1056; Ike etal., 1983, Nucleic Acid Res. 11:477.

In addition, libraries of fragments of the PKSRP coding regions can beused to generate a variegated population of PKSRP fragments forscreening and subsequent selection of homologs of a PKSRP. In oneembodiment, a library of coding sequence fragments can be generated bytreating a double stranded PCR fragment of a PKSRP coding sequence witha nuclease under conditions wherein nicking occurs only about once permolecule, denaturing the double stranded DNA, renaturing the DNA to formdouble stranded DNA, which can include sense/antisense pairs fromdifferent nicked products, removing single stranded portions fromreformed duplexes by treatment with S1 nuclease, and ligating theresulting fragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminal,and internal fragments of various sizes of the PKSRP.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of PKSRP homologs. The mostwidely used techniques, which are amenable to high through-put analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a newtechnique that enhances the frequency of functional mutants in thelibraries, can be used in combination with the screening assays toidentify PKSRP homologs (Arkin and Yourvan, 1992, PNAS 89:7811-7815;Delgrave et al., 1993, Polypeptide Engineering 6(3):327-331). In anotherembodiment, cell based assays can be exploited to analyze a variegatedPKSRP library, using methods well known in the art. The presentinvention further provides a method of identifying a novel PKSRP,comprising (a) raising a specific antibody response to a PKSRP, or afragment thereof, as described herein; (b) screening putative PKSRPmaterial with the antibody, wherein specific binding of the antibody tothe material indicates the presence of a potentially novel PKSRP; and(c) analyzing the bound material in comparison to known PKSRP, todetermine its novelty.

As stated above, the present invention includes PKSRPs and homologsthereof. To determine the percent sequence identity of two amino acidsequences (e.g., one of the sequences of SEQ ID NO:3, SEQ ID NO:6, SEQID NO:9, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ IDNO:30, and a mutant form thereof), the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in the sequence of onepolypeptide for optimal alignment with the other polypeptide or nucleicacid). The amino acid residues at corresponding amino acid positions arethen compared. When a position in one sequence (e.g., one of thesequences of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30) is occupied by thesame amino acid residue as the corresponding position in the othersequence (e.g., a mutant form of the sequence selected from thepolypeptide of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30), then the moleculesare identical at that position. The same type of comparison can be madebetween two nucleic acid sequences.

The percent sequence identity between the two sequences is a function ofthe number of identical positions shared by the sequences (i.e., percentsequence identity=numbers of identical positions/total numbers ofpositions×100). Preferably, the isolated amino acid homologs included inthe present invention are at least about 50-60%, preferably at leastabout 60-70%, and more preferably at least about 70-75%, 75-80%, 80-85%,85-90% or 90-95%, and most preferably at least about 96%, 97%, 98%, 99%or more identical to an entire amino acid sequence shown in SEQ ID NO:3,SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, or SEQ ID NO:30. In yet another embodiment, the isolated aminoacid homologs included in the present invention are at least about50-60%, preferably at least about 60-70%, and more preferably at leastabout 70-75%, 75-80%, 80-85%, 85-90% or 90-95%, and most preferably atleast about 96%, 97%, 98%, 99% or more identical to an entire amino acidsequence encoded by a nucleic acid sequence shown in SEQ ID NO:2, SEQ IDNO:5 SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, or SEQ ID NO:29. In other embodiments, the PKSRP amino acidhomologs have sequence identity over at least 15 contiguous amino acidresidues, more preferably at least 25 contiguous amino acid residues,and most preferably at least 35 contiguous amino acid residues of SEQ IDNO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, or SEQ ID NO:30. In one embodiment of the presentinvention, the homolog has at least about 50-60%, preferably at leastabout 60-70%, more preferably at least about 70-75%, 75-80%, 80-85%,85-90% or 90-95%, and even more preferably at least about 95%, 96%, 97%,98%, 99% or more sequence identity with the kinase domain of PK-3 (aminoacids 87-360 of SEQ ID NO:3) or PK-4 (amino acids 81-281 of SEQ IDNO:6).

In another preferred embodiment, an isolated nucleic acid homolog of theinvention comprises a nucleotide sequence which is at least about50-60%, preferably at least about 60-70%, more preferably at least about70-75%, 75-80%, 80-85%, 85-90% or 90-95%, and even more preferably atleast about 95%, 96%, 97%, 98%, 99% or more identical to a nucleotidesequence shown in SEQ ID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ ID NO:11,SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21,SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29, or to aportion comprising at least 60 consecutive nucleotides thereof. Thepreferable length of sequence comparison for nucleic acids is at least75 nucleotides, more preferably at least 100 nucleotides and mostpreferably the entire length of the coding region.

It is further preferred that the isolated nucleic acid homolog of theinvention encodes a PKSRP, or portion thereof, that is at least 85%identical to an amino acid sequence of SEQ ID NO:3, SEQ ID NO:6, SEQ IDNO:9, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ IDNO:30 and that functions as a modulator of an environmental stressresponse in a plant. In a more preferred embodiment, overexpression ofthe nucleic acid homolog in a plant increases the tolerance of the plantto an environmental stress. In a further preferred embodiment, thenucleic acid homolog encodes a PKSRP that functions as a protein kinase.

For the purposes of the invention, the percent sequence identity betweentwo nucleic acid or polypeptide sequences may be determined using theVector NTI 6.0 (PC) software package (InforMax, 7600 Wisconsin Ave.,Bethesda, Md. 20814). A gap opening penalty of 15 and a gap extensionpenalty of 6.66 are used for determining the percent identity of twonucleic acids. A gap opening penalty of 10 and a gap extension penaltyof 0.1 are used for determining the percent identity of twopolypeptides. All other parameters are set at the default settings. Forpurposes of a multiple alignment (Clustal W algorithm), the gap openingpenalty is 10, and the gap extension penalty is 0.05 with blosum62matrix. It is to be understood that for the purposes of determiningsequence identity when comparing a DNA sequence to an RNA sequence, athymidine nucleotide is equivalent to a uracil nucleotide.

In another aspect, the invention provides an isolated nucleic acidcomprising a polynucleotide that hybridizes to the polynucleotide of SEQID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, or SEQ ID NO:29 under stringent conditions. Moreparticularly, an isolated nucleic acid molecule of the invention is atleast 15 nucleotides in length and hybridizes under stringent conditionsto the nucleic acid molecule comprising a nucleotide sequence of SEQ IDNO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15,SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25,SEQ ID NO:27, or SEQ ID NO:29. In other embodiments, the nucleic acid isat least 30, 50, 100, 250 or more nucleotides in length. Preferably, anisolated nucleic acid homolog of the invention comprises a nucleotidesequence which hybridizes under highly stringent conditions to thenucleotide sequence shown in SEQ ID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29, andfunctions as a modulator of stress tolerance in a plant. In a furtherpreferred embodiment, overexpression of the isolated nucleic acidhomolog in a plant increases a plant's tolerance to an environmentalstress. In an even further preferred embodiment, the isolated nucleicacid homolog encodes a PKSRP that functions as a protein kinase.

As used herein with regard to hybridization for DNA to DNA blot, theterm “stringent conditions” refers to hybridization overnight at 60° C.in 10×Denharts solution, 6×SSC, 0.5% SDS and 100 μg/ml denatured salmonsperm DNA. Blots are washed sequentially at 62° C. for 30 minutes eachtime in 3×SSC/0.1% SDS, followed by 1×SSC/0.1% SDS and finally0.1×SSC/0.1% SDS. As also used herein, “highly stringent conditions”refers to hybridization overnight at 65° C. in 10× Denharts solution,6×SSC, 0.5% SDS and 100 μg/ml denatured salmon sperm DNA. Blots arewashed sequentially at 65° C. for 30 minutes each time in 3×SSC/0.1%SDS, followed by 1×SSC/0.1% SDS and finally 0.1×SSC/0.1% SDS. Methodsfor nucleic acid hybridizations are described in Meinkoth and Wahl,1984, Anal. Biochem. 138:267-284; Ausubel et al. eds, 1995, CurrentProtocols in Molecular Biology, Chapter 2, Greene Publishing andWiley-Interscience, New York; and Tijssen, 1993, Laboratory Techniquesin Biochemistry and Molecular Biology: Hybridization with Nucleic AcidProbes, Part I, Chapter 2, Elsevier, N.Y. Preferably, an isolatednucleic acid molecule of the invention that hybridizes under stringentor highly stringent conditions to a sequence of SEQ ID NO:2, SEQ ID NO:5SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQID NO:29 corresponds to a naturally occurring nucleic acid molecule. Asused herein, a “naturally occurring” nucleic acid molecule refers to anRNA or DNA molecule having a nucleotide sequence that occurs in nature(e.g., encodes a natural polypeptide). In one embodiment, the nucleicacid encodes a naturally occurring Physcomitrella patensi, Brassicanapus, Glycine max, or Oryza sativa PKSRP.

Using the above-described methods, and others known to those of skill inthe art, one of ordinary skill in the art can isolate homologs of thePKSRPs comprising amino acid sequences shown in SEQ ID NO:3, SEQ IDNO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, or SEQ ID NO:30. One subset of these homologs are allelicvariants. As used herein, the term “allelic variant” refers to anucleotide sequence containing polymorphisms that lead to changes in theamino acid sequences of a PKSRP and that exist within a naturalpopulation (e.g., a plant species or variety). Such natural allelicvariations can typically result in 1-5% variance in a PKSRP nucleicacid. Allelic variants can be identified by sequencing the nucleic acidsequence of interest in a number of different plants, which can bereadily carried out by using hybridization probes to identify the samePKSRP genetic locus in those plants. Any and all such nucleic acidvariations and resulting amino acid polymorphisms or variations in aPKSRP that are the result of natural allelic variation and that do notalter the functional activity of a PKSRP, are intended to be within thescope of the invention.

Moreover, nucleic acid molecules encoding PKSRPs from the same or otherspecies such as PKSRP analogs, orthologs, and paralogs, are intended tobe within the scope of the present invention. As used herein, the term“analogs” refers to two nucleic acids that have the same or similarfunction, but that have evolved separately in unrelated organisms. Asused herein, the term “orthologs” refers to two nucleic acids fromdifferent species, but that have evolved from a common ancestral gene byspeciation. Normally, orthologs encode polypeptides having the same orsimilar functions. As also used herein, the term “paralogs” refers totwo nucleic acids that are related by duplication within a genome.Paralogs usually have different functions, but these functions may berelated (Tatusov, R. L. et al., 1997, Science 278(5338):631-637).Analogs, orthologs and paralogs of a naturally occurring PKSRP candiffer from the naturally occurring PKSRP by post-translationalmodifications, by amino acid sequence differences, or by both.Post-translational modifications include in vivo and in vitro chemicalderivatization of polypeptides, e.g., acetylation, carboxylation,phosphorylation, or glycosylation, and such modifications may occurduring polypeptide synthesis or processing or following treatment withisolated modifying enzymes. In particular, orthologs of the inventionwill generally exhibit at least 80-85%, more preferably, 85-90% or90-95%, and most preferably 95%, 96%, 97%, 98% or even 99% identity orsequence identity with all or part of a naturally occurring PKSRP aminoacid sequence and will exhibit a function similar to a PKSRP.Preferably, a PKSRP ortholog of the present invention functions as amodulator of an environmental stress response in a plant and/orfunctions as a protein kinase. More preferably, a PKSRP orthologincreases the stress tolerance of a plant. In one embodiment, the PKSRPorthologs maintain the ability to participate in the metabolism ofcompounds necessary for the construction of cellular membranes in aplant, or in the transport of molecules across these membranes.

In addition to naturally-occurring variants of a PKSRP sequence that mayexist in the population, the skilled artisan will further appreciatethat changes can be introduced by mutation into a nucleotide sequence ofSEQ ID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, or SEQ ID NO:29, thereby leading to changes in theamino acid sequence of the encoded PKSRP, without altering thefunctional activity of the PKSRP. For example, nucleotide substitutionsleading to amino acid substitutions at “non-essential” amino acidresidues can be made in a sequence of SEQ ID NO:2, SEQ ID NO:5 SEQ IDNO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQ IDNO:29. A “non-essential” amino acid residue is a residue that can bealtered from the wild-type sequence of one of the PKSRPs withoutaltering the activity of said PKSRP, whereas an “essential” amino acidresidue is required for PKSRP activity. Other amino acid residues,however, (e.g., those that are not conserved or only semi-conserved inthe domain having PKSRP activity) may not be essential for activity andthus are likely to be amenable to alteration without altering PKSRPactivity.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding PKSRPs that contain changes in amino acid residuesthat are not essential for PKSRP activity. Such PKSRPs differ in aminoacid sequence from a sequence contained in SEQ ID NO:3, SEQ ID NO:6, SEQID NO:9, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ IDNO:30, yet retain at least one of the PKSRP activities described herein.In one embodiment, the isolated nucleic acid molecule comprises anucleotide sequence encoding a polypeptide, wherein the polypeptidecomprises an amino acid sequence at least about 50% identical to anamino acid sequence of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30.Preferably, the polypeptide encoded by the nucleic acid molecule is atleast about 50-60% identical to one of the sequences of SEQ ID NO:3, SEQID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, or SEQ ID NO:30, more preferably at least about 60-70% identicalto one of the sequences of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30, evenmore preferably at least about 70-75%, 75-80%, 80-85%, 85-90%, 90-95%identical to one of the sequences of SEQ ID NO:3, SEQ ID NO:6, SEQ IDNO:9, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ IDNO:30, and most preferably at least about 96%, 97%, 98%, or 99%identical to one of the sequences of SEQ ID NO:3, SEQ ID NO:6, SEQ IDNO:9, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ IDNO:30. The preferred PKSRP homologs of the present invention participatein the a stress tolerance response in a plant, or more particularly,participate in the transcription of a polypeptide involved in a stresstolerance response in a plant, and/or function as a protein kinase.

An isolated nucleic acid molecule encoding a PKSRP having sequenceidentity with a polypeptide sequence of SEQ ID NO:3, SEQ ID NO:6, SEQ IDNO:9, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ IDNO:30 can be created by introducing one or more nucleotidesubstitutions, additions or deletions into a nucleotide sequence of SEQID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ ID NO:1, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, or SEQ ID NO:29, respectively, such that one ormore amino acid substitutions, additions, or deletions are introducedinto the encoded polypeptide. Mutations can be introduced into one ofthe sequences of SEQ ID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ ID NO:11, SEQID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29 by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Preferably, conservative amino acid substitutions are madeat one or more predicted non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain.

Families of amino acid residues having similar side chains have beendefined in the art. These families include amino acids with basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a PKSRP is preferablyreplaced with another amino acid residue from the same side chainfamily. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of a PKSRP coding sequence, suchas by saturation mutagenesis, and the resultant mutants can be screenedfor a PKSRP activity described herein to identify mutants that retainPKSRP activity. Following mutagenesis of one of the sequences of SEQ IDNO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15,SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25,SEQ ID NO:27, or SEQ ID NO:29, the encoded polypeptide can be expressedrecombinantly and the activity of the polypeptide can be determined byanalyzing the stress tolerance of a plant expressing the polypeptide asdescribed in Example 7.

Additionally, optimized PKSRP nucleic acids can be created. Preferably,an optimized PKSRP nucleic acid encodes a PKSRP that functions as aprotein kinase and/or modulates a plant's tolerance to an environmentalstress, and more preferably increases a plant's tolerance to anenvironmental stress upon its overexpression in the plant. As usedherein, “optimized” refers to a nucleic acid that is geneticallyengineered to increase its expression in a given plant or animal. Toprovide plant optimized PKSRP nucleic acids, the DNA sequence of thegene can be modified to 1) comprise codons preferred by highly expressedplant genes; 2) comprise an A+T content in nucleotide base compositionto that substantially found in plants; 3) form a plant initiationsequence; or 4) eliminate sequences that cause destabilization,inappropriate polyadenylation, degradation, and termination of RNA, orthat form secondary structure hairpins or RNA splice sites. Increasedexpression of PKSRP nucleic acids in plants can be achieved by utilizingthe distribution frequency of codon usage in plants in general or aparticular plant. Methods for optimizing nucleic acid expression inplants can be found in EPA 0359472; EPA 0385962; PCT Application No. WO91/16432; U.S. Pat. No. 5,380,831; U.S. Pat. No. 5,436,391; Perlack etal., 1991, Proc. Natl. Acad. Sci. USA 88:3324-3328; and Murray et al.,1989, Nucleic Acids Res. 17:477-498.

As used herein, “frequency of preferred codon usage” refers to thepreference exhibited by a specific host cell in usage of nucleotidecodons to specify a given amino acid. To determine the frequency ofusage of a particular codon in a gene, the number of occurrences of thatcodon in the gene is divided by the total number of occurrences of allcodons specifying the same amino acid in the gene. Similarly, thefrequency of preferred codon usage exhibited by a host cell can becalculated by averaging frequency of preferred codon usage in a largenumber of genes expressed by the host cell. It is preferable that thisanalysis be limited to genes that are highly expressed by the host cell.The percent deviation of the frequency of preferred codon usage for asynthetic gene from that employed by a host cell is calculated first bydetermining the percent deviation of the frequency of usage of a singlecodon from that of the host cell followed by obtaining the averagedeviation over all codons. As defined herein, this calculation includesunique codons (i.e., ATG and TGG). In general terms, the overall averagedeviation of the codon usage of an optimized gene from that of a hostcell is calculated using the equation 1A=n=1 Z X_(n)−Y_(n)X_(n) times100 Z where X_(n)=frequency of usage for codon n in the host cell;Y_(n)=frequency of usage for codon n in the synthetic gene; n representsan individual codon that specifies an amino acid; and the total numberof codons is Z. The overall deviation of the frequency of codon usage,A, for all amino acids should preferably be less than about 25%, andmore preferably less than about 10%.

Hence, a PKSRP nucleic acid can be optimized such that its distributionfrequency of codon usage deviates, preferably, no more than 25% fromthat of highly expressed plant genes and, more preferably, no more thanabout 10%. In addition, consideration is given to the percentage G+Ccontent of the degenerate third base (monocotyledons appear to favor G+Cin this position, whereas dicotyledons do not). It is also recognizedthat the XCG (where X is A, T, C, or G) nucleotide is the leastpreferred codon in dicots whereas the XTA codon is avoided in bothmonocots and dicots. Optimized PKSRP nucleic acids of this inventionalso preferably have CG and TA doublet avoidance indices closelyapproximating those of the chosen host plant (i.e., Physcomitrellapatens, Brassica napits, Glycine max, or Oryza sativa). More preferablythese indices deviate from that of the host by no more than about10-15%.

In addition to the nucleic acid molecules encoding the PKSRPs describedabove, another aspect of the invention pertains to isolated nucleic acidmolecules that are antisense thereto. Antisense polynucleotides arethought to inhibit gene expression of a target polynucleotide byspecifically binding the target polynucleotide and interfering withtranscription, splicing, transport, translation, and/or stability of thetarget polynucleotide. Methods are described in the prior art fortargeting the antisense polynucleotide to the chromosomal DNA, to aprimary RNA transcript, or to a processed mRNA. Preferably, the targetregions include splice sites, translation initiation codons, translationtermination codons, and other sequences within the open reading frame.

The term “antisense,” for the purposes of the invention, refers to anucleic acid comprising a polynucleotide that is sufficientlycomplementary to all or a portion of a gene, primary transcript, orprocessed mRNA, so as to interfere with expression of the endogenousgene. “Complementary” polynucleotides are those that are capable of basepairing according to the standard Watson-Crick complementarity rules.Specifically, purines will base pair with pyrimidines to form acombination of guanine paired with cytosine (G:C) and adenine pairedwith either thymine (A:T) in the case of DNA, or adenine paired withuracil (A:U) in the case of RNA. It is understood that twopolynucleotides may hybridize to each other even if they are notcompletely complementary to each other, provided that each has at leastone region that is substantially complementary to the other. The term“antisense nucleic acid” includes single stranded RNA as well asdouble-stranded DNA expression cassettes that can be transcribed toproduce an antisense RNA. “Active” antisense nucleic acids are antisenseRNA molecules that are capable of selectively hybridizing with a primarytranscript or mRNA encoding a polypeptide having at least 80% sequenceidentity with the polypeptide of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9,SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20,SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30.

The antisense nucleic acid can be complementary to an entire PKSRPcoding strand, or to only a portion thereof. In one embodiment, anantisense nucleic acid molecule is antisense to a “coding region” of thecoding strand of a nucleotide sequence encoding a PKSRP. The term“coding region” refers to the region of the nucleotide sequencecomprising codons that are translated into amino acid residues (e.g.,the entire coding region of PK-3 comprises nucleotides 138-1409 of SEQID NO:2, and the entire coding region of PK-4 comprises nucleotides142-1395 of SEQ ID NO:5). In another embodiment, the antisense nucleicacid molecule is antisense to a “noncoding region” of the coding strandof a nucleotide sequence encoding a PKSRP. The term “noncoding region”refers to 5′ and 3′ sequences that flank the coding region that are nottranslated into amino acids (i.e., also referred to as 5′ and 3′untranslated regions). The antisense nucleic acid molecule can becomplementary to the entire coding region of PKSRP mRNA, but morepreferably is an oligonucleotide which is antisense to only a portion ofthe coding or noncoding region of PKSRP mRNA. For example, the antisenseoligonucleotide can be complementary to the region surrounding thetranslation start site of PKSRP mRNA. An antisense oligonucleotide canbe, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50nucleotides in length. Typically, the antisense molecules of the presentinvention comprise an RNA having 60-100% sequence identity with at least14 consecutive nucleotides of SEQ ID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29 or apolynucleotide encoding SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30.Preferably, the sequence identity will be at least 70%, more preferablyat least 75%, 80%, 85%, 90%, 95%, 98% and most preferably 99%.

An antisense nucleic acid of the invention can be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. For example, an antisense nucleic acid (e.g., anantisense oligonucleotide) can be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed between the antisense and sensenucleic acids, e.g., phosphorothioate derivatives and acridinesubstituted nucleotides can be used. Examples of modified nucleotideswhich can be used to generate the antisense nucleic acid include5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al., 1987, Nucleic Acids. Res. 15:6625-6641).The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al., 1987, Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

The antisense nucleic acid molecules of the invention are typicallyadministered to a cell or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a PKSRP tothereby inhibit expression of the polypeptide, e.g., by inhibitingtranscription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. The antisense molecule can be modified such that itspecifically binds to a receptor or an antigen expressed on a selectedcell surface, e.g., by linking the antisense nucleic acid molecule to apeptide or an antibody which binds to a cell surface receptor orantigen. The antisense nucleic acid molecule can also be delivered tocells using the vectors described herein. To achieve sufficientintracellular concentrations of the antisense molecules, vectorconstructs in which the antisense nucleic acid molecule is placed underthe control of a strong prokaryotic, viral, or eukaryotic (includingplant) promoter are preferred.

As an alternative to antisense polynucleotides, ribozymes, sensepolynucleotides, or double stranded RNA (dsRNA) can be used to reduceexpression of a PKSRP polypeptide. By “ribozyme” is meant a catalyticRNA-based enzyme with ribonuclease activity which is capable of cleavinga single-stranded nucleic acid, such as an mRNA, to which it has acomplementary region. Ribozymes (e.g., hammerhead ribozymes described inHaselhoff and Gerlach, 1988, Nature 334:585-591) can be used tocatalytically cleave PKSRP mRNA transcripts to thereby inhibittranslation of PKSRP mRNA. A ribozyme having specificity for aPKSRP-encoding nucleic acid can be designed based upon the nucleotidesequence of a PKSRP cDNA, as disclosed herein (i.e., SEQ ID NO:2, SEQ IDNO:5 SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, or SEQ ID NO:29) or on the basis of a heterologous sequence to beisolated according to methods taught in this invention. For example, aderivative of a Tetrahymena L-19 IVS RNA can be constructed in which thenucleotide sequence of the active site is complementary to thenucleotide sequence to be cleaved in a PKSRP-encoding mRNA. See, e.g.,U.S. Pat. Nos. 4,987,071 and 5,116,742 to Cech et al. Alternatively,PKSRP mRNA can be used to select a catalytic RNA having a specificribonuclease activity from a pool of RNA molecules. See, e.g., Bartel,D. and Szostak, J. W., 1993, Science 261:1411-1418. In preferredembodiments, the ribozyme will contain a portion having at least 7, 8,9, 10, 12, 14, 16, 18 or 20 nucleotides, and more preferably 7 or 8nucleotides, that have 100% complementarity to a portion of the targetRNA. Methods for making ribozymes are known to those skilled in the art.See, e.g., U.S. Pat. Nos. 6,025,167; 5,773,260; and 5,496,698.

The term “dsRNA,” as used herein, refers to RNA hybrids comprising twostrands of RNA. The dsRNAs can be linear or circular in structure. In apreferred embodiment, dsRNA is specific for a polynucleotide encodingeither the polypeptide of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30 or apolypeptide having at least 70% sequence identity with SEQ ID NO:3, SEQID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, or SEQ ID NO:30. The hybridizing RNAs may be substantially orcompletely complementary. By “substantially complementary,” is meantthat when the two hybridizing RNAs are optimally aligned using the BLASTprogram as described above, the hybridizing portions are at least 95%complementary. Preferably, the dsRNA will be at least 100 base pairs inlength. Typically, the hybridizing RNAs will be of identical length withno over hanging 5′ or 3′ ends and no gaps. However, dsRNAs having 5′ or3′ overhangs of up to 100 nucleotides may be used in the methods of theinvention.

The dsRNA may comprise ribonucleotides or ribonucleotide analogs, suchas 2′-O-methyl ribosyl residues, or combinations thereof. See, e.g.,U.S. Pat. Nos. 4,130,641 and 4,024,222. A dsRNA polyriboinosinicacid:polyribocytidylic acid is described in U.S. Pat. No. 4,283,393.Methods for making and using dsRNA are known in the art. One methodcomprises the simultaneous transcription of two complementary DNAstrands, either in vivo, or in a single in vitro reaction mixture. See,e.g., U.S. Pat. No. 5,795,715. In one embodiment, dsRNA can beintroduced into a plant or plant cell directly by standardtransformation procedures. Alternatively, dsRNA can be expressed in aplant cell by transcribing two complementary RNAs.

Other methods for the inhibition of endogenous gene expression, such astriple helix formation (Moser et al., 1987, Science 238:645-650 andCooney et al., 1988, Science 241:456-459) and cosuppression (Napoli etal., 1990, The Plant Cell 2:279-289) are known in the art. Partial andfull-length cDNAs have been used for the cosuppression of endogenousplant genes. See, e.g., U.S. Pat. Nos. 4,801,340, 5,034,323, 5,231,020,and 5,283,184; Van der Kroll et al., 1990, The Plant Cell 2:291-299;Smith et al., 1990, Mol. Gen. Genetics 224:477-481 and Napoli et al.,1990, The Plant Cell 2:279-289.

For sense suppression, it is believed that introduction of a sensepolynucleotide blocks transcription of the corresponding target gene.The sense polynucleotide will have at least 65% sequence identity withthe target plant gene or RNA. Preferably, the percent identity is atleast 80%, 90%, 95% or more. The introduced sense polynucleotide neednot be full length relative to the target gene or transcript.Preferably, the sense polynucleotide will have at least 65% sequenceidentity with at least 100 consecutive nucleotides of SEQ ID NO:2, SEQID NO:5 SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, or SEQ ID NO:29. The regions of identity can comprise introns andand/or exons and untranslated regions. The introduced sensepolynucleotide may be present in the plant cell transiently, or may bestably integrated into a plant chromosome or extrachromosomal replicon.

Alternatively, PKSRP gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of a PKSRPnucleotide sequence (e.g., a PKSRP promoter and/or enhancer) to formtriple helical structures that prevent transcription of a PKSRP gene intarget cells. See generally, Helene, C., 1991, Anticancer Drug Des.6(6):569-84; Helene, C. et al., 1992, Ann. N.Y. Acad. Sci. 660:27-36;and Maher, L. J., 1992, Bioassays 14(12):807-15.

In addition to the PKSRP nucleic acids and polypeptides described above,the present invention encompasses these nucleic acids and polypeptidesattached to a moiety. These moieties include, but are not limited to,detection moieties, hybridization moieties, purification moieties,delivery moieties, reaction moieties, binding moieties, and the like. Atypical group of nucleic acids having moieties attached are probes andprimers. Probes and primers typically comprise a substantially isolatedoligonucleotide. The oligonucleotide typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, preferably about 25, more preferably about 40, 50 or 75consecutive nucleotides of a sense strand of one of the sequences setforth in SEQ ID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ ID NO:1, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29; an anti-sensesequence of one of the sequences set forth in SEQ ID NO:2, SEQ ID NO:5SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQID NO:29; or naturally occurring mutants thereof. Primers based on anucleotide sequence of SEQ ID NO:2, SEQ ID NO:5 SEQ ID NO:8, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, or SEQ ID NO:29 can beused in PCR reactions to clone PKSRP homologs. Probes based on the PKSRPnucleotide sequences can be used to detect transcripts or genomicsequences encoding the same or substantially identical polypeptides. Inpreferred embodiments, the probe further comprises a label groupattached thereto, e.g. the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as a part of a genomic marker test kit for identifying cellswhich express a PKSRP, such as by measuring a level of a PKSRP-encodingnucleic acid, in a sample of cells, e.g., detecting PKSRP mRNA levels ordetermining whether a genomic PKSRP gene has been mutated or deleted.

In particular, a useful method to ascertain the level of transcriptionof the gene (an indicator of the amount of mRNA available fortranslation to the gene product) is to perform a Northern blot. Forreference, see, for example, Ausubel et al., 1988, Current Protocols inMolecular Biology, Wiley: New York. The information from a Northern blotat least partially demonstrates the degree of transcription of thetransformed gene. Total cellular RNA can be prepared from cells, tissuesor organs by several methods, all well-known in the art, such as thatdescribed in Bormann, E. R. et al., 1992, Mol. Microbiol. 6:317-326. Toassess the presence or relative quantity of polypeptide translated fromthis mRNA, standard techniques, such as a Western blot, may be employed.These techniques are well known to one of ordinary skill in the art.See, for example, Ausubel et al., 1988, Current Protocols in MolecularBiology, Wiley: New York.

The invention further provides an isolated recombinant expression vectorcomprising a PKSRP nucleic acid as described above, wherein expressionof the vector in a host cell results in increased tolerance toenvironmental stress as compared to a wild type variety of the hostcell. As used herein, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors.” In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses, and adeno-associatedviruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. As used herein with respect to arecombinant expression vector, “operatively linked” is intended to meanthat the nucleotide sequence of interest is linked to the regulatorysequence(s) in a manner which allows for expression of the nucleotidesequence (e.g., in an in vitro transcription/translation system or in ahost cell when the vector is introduced into the host cell). The term“regulatory sequence” is intended to include promoters, enhancers, andother expression control elements (e.g., polyadenylation signals). Suchregulatory sequences are described, for example, in Goeddel, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) and Gruber and Crosby, in: Methods in PlantMolecular Biology and Biotechnology, eds. Glick and Thompson, Chapter 7,89-108, CRC Press: Boca Raton, Fla., including the references therein.Regulatory sequences include those that direct constitutive expressionof a nucleotide sequence in many types of host cells and those thatdirect expression of the nucleotide sequence only in certain host cellsor under certain conditions. It will be appreciated by those skilled inthe art that the design of the expression vector can depend on suchfactors as the choice of the host cell to be transformed, the level ofexpression of polypeptide desired, etc. The expression vectors of theinvention can be introduced into host cells to thereby producepolypeptides or peptides, including fusion polypeptides or peptides,encoded by nucleic acids as described herein (e.g., PKSRPs, mutant formsof PKSRPs, fusion polypeptides, etc.).

The recombinant expression vectors of the invention can be designed forexpression of PKSRPs in prokaryotic or eukaryotic cells. For example,PKSRP genes can be expressed in bacterial cells such as C. glutamicum,insect cells (using baculovirus expression vectors), yeast and otherfungal cells (See Romanos, M. A. et al., 1992, Foreign gene expressionin yeast: a review, Yeast 8:423-488; van den Hondel, C. A. M. J. J. etal., 1991, Heterologous gene expression in filamentous fungi, in: MoreGene Manipulations in Fungi, J. W. Bennet & L. L. Lasure, eds., p.396-428: Academic Press: San Diego; and van den Hondel, C. A. M. J. J. &Punt, P. J., 1991, Gene transfer systems and vector development forfilamentous fungi, in: Applied Molecular Genetics of Fungi, Peberdy, J.F. et al., eds., p. 1-28, Cambridge University Press: Cambridge), algae(Falciatore et al., 1999, Marine Biotechnology 1(3):239-251), ciliatesof the types: Holotrichia, Peritrichia, Spirotrichia, Suctoria,Tetrahymena, Paramecium, Colpidium, Glaucoma, Platyophrya, Potomacus,Pseudocohnilembus, Euplotes, Engelmaniella, and Stylonychia, especiallyof the genus Stylonychia lemnae with vectors following a transformationmethod as described in PCT Application No. WO 98/01572, andmulticellular plant cells (See Schmidt, R. and Willmitzer, L., 1988,High efficiency Agrobacterium tumefaciens-mediated transformation ofArabidopsis thaliana leaf and cotyledon explants, Plant Cell Rep.583-586; Plant Molecular Biology and Biotechnology, C Press, Boca Raton,Fla., chapter 6/7, S.71-119 (1993); F. F. White, B. Jenes et al.,Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineeringand Utilization, eds. Kung und R. Wu, 128-43, Academic Press: 1993;Potrykus, 1991, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42:205-225and references cited therein) or mammalian cells. Suitable host cellsare discussed further in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press: San Diego, Calif. (1990). Alternatively,the recombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Expression of polypeptides in prokaryotes is most often carried out withvectors containing constitutive or inducible promoters directing theexpression of either fusion or non-fusion polypeptides. Fusion vectorsadd a number of amino acids to a polypeptide encoded therein, usually tothe amino terminus of the recombinant polypeptide but also to theC-terminus or fused within suitable regions in the polypeptides. Suchfusion vectors typically serve three purposes: 1) to increase expressionof a recombinant polypeptide; 2) to increase the solubility of arecombinant polypeptide; and 3) to aid in the purification of arecombinant polypeptide by acting as a ligand in affinity purification.Often, in fusion expression vectors, a proteolytic cleavage site isintroduced at the junction of the fusion moiety and the recombinantpolypeptide to enable separation of the recombinant polypeptide from thefusion moiety subsequent to purification of the fusion polypeptide. Suchenzymes, and their cognate recognition sequences, include Factor Xa,thrombin, and enterokinase.

Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S., 1988, Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding polypeptide, orpolypeptide A, respectively, to the target recombinant polypeptide. Inone embodiment, the coding sequence of the PKSRP is cloned into a pGEXexpression vector to create a vector encoding a fusion polypeptidecomprising, from the N-terminus to the C-terminus, GST-thrombin cleavagesite-X polypeptide. The fusion polypeptide can be purified by affinitychromatography using glutathione-agarose resin. Recombinant PKSRPunfused to GST can be recovered by cleavage of the fusion polypeptidewith thrombin.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., 1988, Gene 69:301-315) and pET 11d (Studieret al., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET 11d vectorrelies on transcription from a T7 gn10-lac fusion promoter mediated by aco-expressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident λprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant polypeptide expression is toexpress the polypeptide in a host bacteria with an impaired capacity toproteolytically cleave the recombinant polypeptide (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the sequenceof the nucleic acid to be inserted into an expression vector so that theindividual codons for each amino acid are those preferentially utilizedin the bacterium chosen for expression, such as C. glutamicum (Wada etal., 1992, Nucleic Acids Res. 20:2111-2118). Such alteration of nucleicacid sequences of the invention can be carried out by standard DNAsynthesis techniques.

In another embodiment, the PKSRP expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari, et al., 1987, EMBO J. 6:229-234), pMFa(Kurjan and Herskowitz, 1982, Cell 30:933-943), pJRY88 (Schultz et al.,1987, Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego,Calif.). Vectors and methods for the construction of vectors appropriatefor use in other fungi, such as the filamentous fungi, include thosedetailed in: van den Hondel, C. A. M. J. J. & Punt, P. J., 1991, “Genetransfer systems and vector development for filamentous fungi,” in:Applied Molecular Genetics of Fungi, J. F. Peberdy, et al., eds., p.1-28, Cambridge University Press: Cambridge.

Alternatively, the PKSRPs of the invention can be expressed in insectcells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of polypeptides in cultured insect cells (e.g.,Sf 9 cells) include the pAc series (Smith et al., 1983, Mol. Cell. Biol.3:2156-2165) and the pVL series (Lucklow and Summers, 1989, Virology170:31-39).

In yet another embodiment, a PKSRP nucleic acid of the invention isexpressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, B., 1987,Nature 329:840) and pMT2PC (Kaufman et al., 1987, EMBO J. 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirus,and Simian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells, see chapters 16 and 17 of Sambrook,J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A LaboratoryManual. 2^(nd), ed., Cold Spring Harbor Laboratory, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.,1987, Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton, 1988, Adv. Immunol. 43:235-275), in particular promoters of Tcell receptors (Winoto and Baltimore, 1989, EMBO J. 8:729-733) andimmunoglobulins (Banerji et al., 1983, Cell 33:729-740; Queen andBaltimore, 1983, Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989, PNAS 86:5473-5477),pancreas-specific promoters (Edlund et al., 1985, Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally-regulated promoters are also encompassed, for example,the murine hox promoters (Kessel and Gruss, 1990, Science 249:374-379)and the fetopolypeptide promoter (Campes and Tilghman, 1989, Genes Dev.3:537-546).

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin, and methotrexate, or in plants thatconfer resistance towards a herbicide such as glyphosate or glufosinate.Nucleic acid molecules encoding a selectable marker can be introducedinto a host cell on the same vector as that encoding a PKSRP or can beintroduced on a separate vector. Cells stably transfected with theintroduced nucleic acid molecule can be identified by, for example, drugselection (e.g., cells that have incorporated the selectable marker genewill survive, while the other cells die).

In a preferred embodiment of the present invention, the PKSRPs areexpressed in plants and plants cells such as unicellular plant cells(e.g. algae) (See Falciatore et al., 1999, Marine Biotechnology1(3):239-251 and references therein) and plant cells from higher plants(e.g., the spermatophytes, such as crop plants). A PKSRP may be“introduced” into a plant cell by any means, including transfection,transformation or transduction, electroporation, particle bombardment,agroinfection, and the like. One transformation method known to those ofskill in the art is the dipping of a flowering plant into anAgrobacteria solution, wherein the Agrobacteria contains the PKSRPnucleic acid, followed by breeding of the transformed gametes.

Other suitable methods for transforming or transfecting host cellsincluding plant cells can be found in Sambrook, et al., MolecularCloning: A Laboratory Manual. 2^(nd), ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989, and other laboratory manuals such as Methods in MolecularBiology, 1995, Vol. 44, Agrobacterium protocols, ed: Gartland and Davey,Humana Press, Totowa, N.J. As biotic and abiotic stress tolerance is ageneral trait wished to be inherited into a wide variety of plants likemaize, wheat, rye, oat, triticale, rice, barley, soybean, peanut,cotton, rapeseed and canola, manihot, pepper, sunflower and tagetes,solanaceous plants like potato, tobacco, eggplant, and tomato, Viciaspecies, pea, alfalfa, bushy plants (coffee, cacao, tea), Salix species,trees (oil palm, coconut), perennial grasses, and forage crops, thesecrop plants are also preferred target plants for a genetic engineeringas one further embodiment of the present invention. Forage cropsinclude, but are not limited to, Wheatgrass, Canarygrass, Bromegrass,Wildrye Grass, Bluegrass, Orchardgrass, Alfalfa, Salfoin, BirdsfootTrefoil, Alsike Clover, Red Clover, and Sweet Clover.

In one embodiment of the present invention, transfection of a PKSRP intoa plant is achieved by Agrobacterium mediated gene transfer.Agrobacterium mediated plant transformation can be performed using forexample the GV3101 (pMP90) (Koncz and Schell, 1986, Mol. Gen. Genet.204:383-396) or LBA4404 (Clontech) Agrobacterium tumefaciens strain.Transformation can be performed by standard transformation andregeneration techniques (Deblaere et al., 1994, Nucl. Acids Res.13:4777-4788; Gelvin, Stanton B. and Schilperoort, Robert A, PlantMolecular Biology Manual, 2^(nd) Ed. —Dordrecht: Kluwer Academic Publ.,1995. —in Sect., Ringbuc Zentrale Signatur: BT11-P ISBN 0-7923-2731-4;Glick, Bernard R.; Thompson, John E., Methods in Plant Molecular Biologyand Biotechnology, Boca Raton: CRC Press, 1993 360 S., ISBN0-8493-5164-2). For example, rapeseed can be transformed via cotyledonor hypocotyl transformation (Moloney et al., 1989, Plant cell Report8:238-242; De Block et al., 1989, Plant Physiol. 91:694-701). Use ofantibiotics for Agrobacterium and plant selection depends on the binaryvector and the Agrobacterium strain used for transformation. Rapeseedselection is normally performed using kanamycin as selectable plantmarker. Agrobacterium mediated gene transfer to flax can be performedusing, for example, a technique described by Mlynarova et al., 1994,Plant Cell Report 13:282-285. Additionally, transformation of soybeancan be performed using for example a technique described in EuropeanPatent No. 0424 047, U.S. Pat. No. 5,322,783, European Patent No. 0397687, U.S. Pat. No. 5,376,543, or U.S. Pat. No. 5,169,770. Transformationof maize can be achieved by particle bombardment, polyethylene glycolmediated DNA uptake or via the silicon carbide fiber technique. (See,for example, Freeling and Walbot “The maize handbook” Springer Verlag:New York (1993) ISBN 3-540-97826-7). A specific example of maizetransformation is found in U.S. Pat. No. 5,990,387, and a specificexample of wheat transformation can be found in PCT Application No. WO93/07256.

According to the present invention, the introduced PKSRP may bemaintained in the plant cell stably if it is incorporated into anon-chromosomal autonomous replicon or integrated into the plantchromosomes. Alternatively, the introduced PKSRP may be present on anextra-chromosomal non-replicating vector and be transiently expressed ortransiently active.

In one embodiment, a homologous recombinant microorganism can be createdwherein the PKSRP is integrated into a chromosome, a vector is preparedwhich contains at least a portion of a PKSRP gene into which a deletion,addition, or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the PKSRP gene. Preferably, the PKSRP gene is aPhyscomitrella patens, Brassica napus, Glycine max, or Oryza sativaPKSRP gene, but it can be a homolog from a related plant or even from amammalian, yeast, or insect source. In one embodiment, the vector isdesigned such that, upon homologous recombination, the endogenous PKSRPgene is functionally disrupted (i.e., no longer encodes a functionalpolypeptide; also referred to as a knock-out vector). Alternatively, thevector can be designed such that, upon homologous recombination, theendogenous PKSRP gene is mutated or otherwise altered but still encodesa functional polypeptide (e.g., the upstream regulatory region can bealtered to thereby alter the expression of the endogenous PKSRP). Tocreate a point mutation via homologous recombination, DNA-RNA hybridscan be used in a technique known as chimeraplasty (Cole-Strauss et al.,1999, Nucleic Acids Research 27(5):1323-1330 and Kmiec, 1999 Genetherapy American Scientist. 87(3):240-247). Homologous recombinationprocedures in Physcomitrella patens are also well known in the art andare contemplated for use herein.

Whereas in the homologous recombination vector, the altered portion ofthe PKSRP gene is flanked at its 5′ and 3′ ends by an additional nucleicacid molecule of the PKSRP gene to allow for homologous recombination tooccur between the exogenous PKSRP gene carried by the vector and anendogenous PKSRP gene, in a microorganism or plant. The additionalflanking PKSRP nucleic acid molecule is of sufficient length forsuccessful homologous recombination with the endogenous gene. Typically,several hundreds of base pairs up to kilobases of flanking DNA (both atthe 5′ and 3′ ends) are included in the vector. See, e.g., Thomas, K.R., and Capecchi, M. R., 1987, Cell 51:503 for a description ofhomologous recombination vectors or Strepp et al., 1998, PNAS, 95(8):4368-4373 for cDNA based recombination in Physcomitrella patens).The vector is introduced into a microorganism or plant cell (e.g., viapolyethylene glycol mediated DNA), and cells in which the introducedPKSRP gene has homologously recombined with the endogenous PKSRP geneare selected using art-known techniques.

In another embodiment, recombinant microorganisms can be produced thatcontain selected systems which allow for regulated expression of theintroduced gene. For example, inclusion of a PKSRP gene on a vectorplacing it under control of the lac operon permits expression of thePKSRP gene only in the presence of IPTG. Such regulatory systems arewell known in the art.

Whether present in an extra-chromosomal non-replicating vector or avector that is integrated into a chromosome, the PKSRP polynucleotidepreferably resides in a plant expression cassette. A plant expressioncassette preferably contains regulatory sequences capable of drivinggene expression in plant cells that are operatively linked so that eachsequence can fulfill its function, for example, termination oftranscription by polyadenylation signals. Preferred polyadenylationsignals are those originating from Agrobacterium tumefaciens t-DNA suchas the gene 3 known as octopine synthase of the Ti-plasmid pTiACH5(Gielen et al., 1984, EMBO J. 3:835) or functional equivalents thereofbut also all other terminators functionally active in plants aresuitable. As plant gene expression is very often not limited ontranscriptional levels, a plant expression cassette preferably containsother operatively linked sequences like translational enhancers such asthe overdrive-sequence containing the 5′-untranslated leader sequencefrom tobacco mosaic virus enhancing the polypeptide per RNA ratio(Gallie et al., 1987, Nucl. Acids Research 15:8693-8711). Examples ofplant expression vectors include those detailed in: Becker, D. et al.,1992, New plant binary vectors with selectable markers located proximalto the left border, Plant Mol. Biol. 20: 1195-1197; and Bevan, M. W.,1984, Binary Agrobacterium vectors for plant transformation, Nucl. Acid.Res. 12:8711-8721; and Vectors for Gene Transfer in Higher Plants; in:Transgenic Plants, Vol. 1, Engineering and Utilization, eds.: Kung andR. Wu, Academic Press, 1993, S. 15-38.

Plant gene expression should be operatively linked to an appropriatepromoter conferring gene expression in a timely, cell or tissue specificmanner. Promoters useful in the expression cassettes of the inventioninclude any promoter that is capable of initiating transcription in aplant cell. Such promoters include, but are not limited to, those thatcan be obtained from plants, plant viruses, and bacteria that containgenes that are expressed in plants, such as Agrobacterium and Rhizobium.

The promoter may be constitutive, inducible, developmentalstage-preferred, cell type-preferred, tissue-preferred, ororgan-preferred. Constitutive promoters are active under mostconditions. Examples of constitutive promoters include the CaMV 19S and35 S promoters (Odell et al., 1985, Nature 313:810-812), the sX CaMV 35Spromoter (Kay et al., 1987, Science 236:1299-1302) the Sep1 promoter,the rice actin promoter (McElroy et al., 1990, Plant Cell 2:163-171),the Arabidopsis actin promoter, the ubiquitan promoter (Christensen etal., 1989, Plant Molec Biol 18:675-689); pEmu (Last et al., 1991, TheorAppl Genet. 81:581-588), the figwort mosaic virus 35S promoter, the Smaspromoter (Velten et al., 1984, EMBO J. 3:2723-2730), the GRP1-8promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No.5,683,439), promoters from the T-DNA of Agrobacterium, such as mannopinesynthase, nopaline synthase, and octopine synthase, the small subunit ofribulose biphosphate carboxylase (ssuRUBISCO) promoter, and the like.

Inducible promoters are active under certain environmental conditions,such as the presence or absence of a nutrient or metabolite, heat orcold, light, pathogen attack, anaerobic conditions, and the like. Forexample, the hsp80 promoter from Brassica is induced by heat shock; thePPDK promoter is induced by light; the PR-1 promoter from tobacco,Arabidopsis, and maize are inducible by infection with a pathogen; andthe Adh1 promoter is induced by hypoxia and cold stress. Plant geneexpression can also be facilitated via an inducible promoter (For areview, see Gatz, 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol.48:89-108). Chemically inducible promoters are especially suitable ifgene expression is wanted to occur in a time specific manner. Examplesof such promoters are a salicylic acid inducible promoter (PCTApplication No. WO 95/19443), a tetracycline inducible promoter (Gatz etal., 1992, Plant J. 2:397-404), and an ethanol inducible promoter (PCTApplication No. WO 93/21334).

In one preferred embodiment of the present invention, the induciblepromoter is a stress-inducible promoter. Stress inducible promotersinclude, but are not limited to, Cor78 (Chak et al., 2000, Planta210:875-883; Hovath et al., 1993, Plant Physiol. 103:1047-1053), Cor15a(Artus et al., 1996, PNAS 93(23):13404-09), Rci2A (Medina et al., 2001,Plant Physiol. 125:1655-66; Nylander et al., 2001, Plant Mol. Biol.45:341-52; Navarre and Goffeau, 2000, EMBO J. 19:2515-24; Capel et al.,1997, Plant Physiol. 115:569-76), Rd22 (Xiong et al., 2001, Plant Cell13:2063-83; Abe et al., 1997, Plant Cell 9:1859-68; Iwasaki et al.,1995, Mol. Gen. Genet. 247:391-8), cDet6 (Lang and Palve, 1992, PlantMol. Biol. 20:951-62), ADH1 (Hoeren et al., 1998, Genetics 149:479-90),KAT1 (Nakamura et al., 1995, Plant Physiol. 109:371-4), KST1(Müller-Röber et al., 1995, EMBO 14:2409-16), Rhal (Terryn et al., 1993,Plant Cell 5:1761-9; Terryn et al., 1992, FEBS Lett. 299(3):287-90),ARSK1 (Atkinson et al., 1997, GenBank Accession #L22302, and PCTApplication No. WO 97/20057), PtxA (Plesch et al., GenBank Accession#X67427), SbHRGP3 (Ahn et al., 1996, Plant Cell 8:1477-90), GH3 (Liu etal., 1994, Plant Cell 6:645-57), the pathogen inducible PRP1-genepromoter (Ward et al., 1993, Plant. Mol. Biol. 22:361-366), the heatinducible hsp80-promoter from tomato (U.S. Pat. No. 5,187,267), coldinducible alpha-amylase promoter from potato (PCT Application No. WO96/12814), or the wound-inducible pinII-promoter (European Patent No.375091). For other examples of drought, cold, and salt-induciblepromoters, such as the RD29A promoter, see Yamaguchi-Shinozalei et al.,1993, Mol. Gen. Genet. 236:331-340.

Developmental stage-preferred promoters are preferentially expressed atcertain stages of development. Tissue and organ preferred promotersinclude those that are preferentially expressed in certain tissues ororgans, such as leaves, roots, seeds, or xylem. Examples of tissuepreferred and organ preferred promoters include, but are not limited tofruit-preferred, ovule-preferred, male tissue-preferred, seed-preferred,integument-preferred, tuber-preferred, stalk-preferred,pericarp-preferred, and leaf-preferred, stigma-preferred,pollen-preferred, anther-preferred, a petal-preferred, sepal-preferred,pedicel-preferred, silique-preferred, stem-preferred, root-preferredpromoters, and the like. Seed preferred promoters are preferentiallyexpressed during seed development and/or germination. For example, seedpreferred promoters can be embryo-preferred, endosperm preferred, andseed coat-preferred. See Thompson et al., 1989, BioEssays 10:108.Examples of seed preferred promoters include, but are not limited to,cellulose synthase (celA), Cim1, gamma-zein, globulin-1, maize 19 kDzein (cZ19B1), and the like.

Other suitable tissue-preferred or organ-preferred promoters include thenapin-gene promoter from rapeseed (U.S. Pat. No. 5,608,152), theUSP-promoter from Vicia faba (Baeumlein et al., 1991, Mol Gen Genet.225(3):459-67), the oleosin-promoter from Arabidopsis (PCT ApplicationNo. WO 98/45461), the phaseolin-promoter from Phaseolus vulgaris (U.S.Pat. No. 5,504,200), the Bce-4-promoter from Brassica (PCT ApplicationNo. WO 91/13980), or the legumin B4 promoter (LeB4; Baeumlein et al.,1992, Plant Journal, 2(2):233-9) as well as promoters conferring seedspecific expression in monocot plants like maize, barley, wheat, rye,rice, etc. Suitable promoters to note are the lpt2 or lpt1-gene promoterfrom barley (PCT Application No. WO 95/15389 and PCT Application No. WO95/23230) or those described in PCT Application No. WO 99/16890(promoters from the barley hordein-gene, rice glutelin gene, rice oryzingene, rice prolamin gene, wheat gliadin gene, wheat glutelin gene, oatglutelin gene, Sorghum kasirin-gene, and rye secalin gene).

Other promoters useful in the expression cassettes of the inventioninclude, but are not limited to, the major chlorophyll a/b bindingprotein promoter, histone promoters, the Ap3 promoter, the β-conglycinpromoter, the napin promoter, the soybean lectin promoter, the maize 15kD zein promoter, the 22 kD zein promoter, the 27 kD zein promoter, theg-zein promoter, the waxy, shrunken 1, shrunken 2 and bronze promoters,the Zm13 promoter (U.S. Pat. No. 5,086,169), the maize polygalacturonasepromoters (PG) (U.S. Pat. Nos. 5,412,085 and 5,545,546), and the SGB6promoter (U.S. Pat. No. 5,470,359), as well as synthetic or othernatural promoters.

Additional flexibility in controlling heterologous gene expression inplants may be obtained by using DNA binding domains and responseelements from heterologous sources (i.e., DNA binding domains fromnon-plant sources). An example of such a heterologous DNA binding domainis the LexA DNA binding domain (Brent and Ptashne, 1985, Cell43:729-736).

The invention further provides a recombinant expression vectorcomprising a PKSRP DNA molecule of the invention cloned into theexpression vector in an antisense orientation. That is, the DNA moleculeis operatively linked to a regulatory sequence in a manner that allowsfor expression (by transcription of the DNA molecule) of an RNA moleculethat is antisense to a PKSRP mRNA. Regulatory sequences operativelylinked to a nucleic acid molecule cloned in the antisense orientationcan be chosen which direct the continuous expression of the antisenseRNA molecule in a variety of cell types. For instance, viral promotersand/or enhancers, or regulatory sequences can be chosen which directconstitutive, tissue specific, or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid, or attenuated virus wherein antisensenucleic acids are produced under the control of a high efficiencyregulatory region. The activity of the regulatory region can bedetermined by the cell type into which the vector is introduced. For adiscussion of the regulation of gene expression using antisense genes,see Weintraub, H. et al., 1986, Antisense RNA as a molecular tool forgenetic analysis, Reviews—Trends in Genetics, Vol. 1(1), and Mol et al.,1990, FEBS Letters 268:427-430.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but they also apply to the progeny or potentialprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein. A host cell can be any prokaryotic or eukaryotic cell. Forexample, a PKSRP can be expressed in bacterial cells such as C.glutamicum, insect cells, fungal cells, or mammalian cells (such asChinese hamster ovary cells (CHO) or COS cells), algae, ciliates, plantcells, fungi, or other microorganisms like C. glutamicum. Other suitablehost cells are known to those skilled in the art.

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) a PKSRP.Accordingly, the invention further provides methods for producing PKSRPsusing the host cells of the invention. In one embodiment, the methodcomprises culturing the host cell of invention (into which a recombinantexpression vector encoding a PKSRP has been introduced, or into whichgenome has been introduced a gene encoding a wild-type or altered PKSRP)in a suitable medium until PKSRP is produced. In another embodiment, themethod further comprises isolating PKSRPs from the medium or the hostcell.

Another aspect of the invention pertains to isolated PKSRPs, andbiologically active portions thereof. An “isolated” or “purified”polypeptide or biologically active portion thereof is free of some ofthe cellular material when produced by recombinant DNA techniques, orchemical precursors or other chemicals when chemically synthesized. Thelanguage “substantially free of cellular material” includes preparationsof PKSRP in which the polypeptide is separated from some of the cellularcomponents of the cells in which it is naturally or recombinantlyproduced. In one embodiment, the language “substantially free ofcellular material” includes preparations of a PKSRP having less thanabout 30% (by dry weight) of non-PKSRP material (also referred to hereinas a “contaminating polypeptide”), more preferably less than about 20%of non-PKSRP material, still more preferably less than about 10% ofnon-PKSRP material, and most preferably less than about 5% non-PKSRPmaterial.

When the PKSRP or biologically active portion thereof is recombinantlyproduced, it is also preferably substantially free of culture medium,i.e., culture medium represents less than about 20%, more preferablyless than about 10%, and most preferably less than about 5% of thevolume of the polypeptide preparation. The language “substantially freeof chemical precursors or other chemicals” includes preparations ofPKSRP in which the polypeptide is separated from chemical precursors orother chemicals that are involved in the synthesis of the polypeptide.In one embodiment, the language “substantially free of chemicalprecursors or other chemicals” includes preparations of a PKSRP havingless than about 30% (by dry weight) of chemical precursors or non-PKSRPchemicals, more preferably less than about 20% chemical precursors ornon-PKSRP chemicals, still more preferably less than about 10% chemicalprecursors or non-PKSRP chemicals, and most preferably less than about5% chemical precursors or non-PKSRP chemicals. In preferred embodiments,isolated polypeptides, or biologically active portions thereof, lackcontaminating polypeptides from the same organism from which the PKSRPis derived. Typically, such polypeptides are produced by recombinantexpression of, for example, a Physcomitrella patens, Brassica napus,Glycine max, or Oryza sativa PKSRP in plants other than Physcomitrellapatens, Brassica napus, Glycine max, or Oryza sativa, or microorganismssuch as C. glutamicum, ciliates, algae or fungi.

The nucleic acid molecules, polypeptides, polypeptide homologs, fusionpolypeptides, primers, vectors, and host cells described herein can beused in one or more of the following methods: identification ofPhyscomitrella patens, Brassica napus, Glycine max, or Oryza sativa andrelated organisms; mapping of genomes of organisms related toPhyscomitrella patens, Brassica napus, Glycine max, or Oryza sativa;identification and localization of Physcomitrella patens, Brassicanapus, Glycine max, or Oryza sativa sequences of interest; evolutionarystudies; determination of PKSRP regions required for function;modulation of a PKSRP activity; modulation of the metabolism of one ormore cell functions; modulation of the transmembrane transport of one ormore compounds; modulation of stress resistance; and modulation ofexpression of PKSRP nucleic acids.

The moss Physcomitrella patens represents one member of the mosses. Itis related to other mosses such as Ceratodon purpureus which is capableof growth in the absence of light. Mosses like Ceratodon andPhyscomitrella share a high degree of sequence identity on the DNAsequence and polypeptide level allowing the use of heterologousscreening of DNA molecules with probes evolving from other mosses ororganisms, thus enabling the derivation of a consensus sequence suitablefor heterologous screening or functional annotation and prediction ofgene functions in third species. The ability to identify such functionscan therefore have significant relevance, e.g., prediction of substratespecificity of enzymes. Further, these nucleic acid molecules may serveas reference points for the mapping of moss genomes, or of genomes ofrelated organisms.

The PKSRP nucleic acid molecules of the invention have a variety ofuses. Most importantly, the nucleic acid and amino acid sequences of thepresent invention can be used to transform plants, thereby inducingtolerance to stresses such as drought, high salinity and cold orlodging. The present invention therefore provides a transgenic planttransformed by a PKSRP nucleic acid, wherein expression of the nucleicacid sequence in the plant results in increased tolerance toenvironmental stress or increased resistance to lodging as compared to awild type variety of the plant. The transgenic plant can be a monocot ora dicot. The invention further provides that the transgenic plant can beselected from maize, wheat, rye, oat, triticale, rice, barley, soybean,peanut, cotton, rapeseed, canola, manihot, pepper, sunflower, tagetes,solanaceous plants, potato, tobacco, eggplant, tomato, Vicia species,pea, alfalfa, coffee, cacao, tea, Salix species, oil palm, coconut,perennial grass, and forage crops, for example.

In particular, the present invention describes using the expression ofPK-3, PK-4, PK-10, and PK-11 of Physcomitrella patens; the BnPK-1,BnPK-2, BnPK-3, and BnPK-4 of Brassica napus; the GmPK-1, GmPK-2,GmPK-3, and GmPK-4 of Glycine max; and the OsPK-1 of Oryza sativa toengineer drought-tolerant, salt-tolerant, cold-tolerant, and/orlodging-resistant plants. This strategy has herein been demonstrated forArabidopsis thaliana, Rapeseed/Canola, soybeans, corn, and wheat, butits application is not restricted to these plants. Accordingly, theinvention provides a transgenic plant containing a PKSRP such as PK-3 asdefined in SEQ ID NO:3, PK-4 as defined in SEQ ID NO:6, PK-10 as definedin SEQ ID NO:9, PK-11 as defined in SEQ ID NO:12, BnPK-1 as defined inSEQ ID NO:14, BnPK-2 as defined in SEQ ID NO:16, BnPK-3 as defined inSEQ ID NO:18, BnPK-4 as defined in SEQ ID NO:20, GmPK-1 as defined inSEQ ID NO:22, GmPK-2 as defined in SEQ ID NO:24, GmPK-3 as defined inSEQ ID NO:26, GmPK-4 as defined in SEQ ID NO:28, and OsPK-1 as definedin SEQ ID NO:30, wherein the plant has an increased tolerance to anenvironmental stress selected from drought, increased salt, decreased orincreased temperature, or lodging. In preferred embodiments, theenvironmental stress is drought or decreased temperature.

Accordingly, the invention provides a method of producing a transgenicplant with a PKSRP coding nucleic acid, wherein expression of thenucleic acid in the plant results in increased tolerance toenvironmental stress as compared to a wild type variety of the plantcomprising: (a) introducing into a plant cell an expression vectorcomprising a PKSRP nucleic acid, and (b) generating from the plant cella transgenic plant with an increased tolerance to environmental stressas compared to a wild type variety of the plant. Also included withinthe present invention are methods of increasing a plant's resistance tolodging, comprising transforming a plant cell with an expressioncassette comprising a nucleic acid encoding a PKSRP and generating atransgenic plant from the transformed plant cell. The plant cellincludes, but is not limited to, a protoplast, gamete producing cell,and a cell that regenerates into a whole plant. As used herein, the term“transgenic” refers to any plant, plant cell, callus, plant tissue, orplant part, that contains all or part of at least one recombinantpolynucleotide. In many cases, all or part of the recombinantpolynucleotide is stably integrated into a chromosome or stableextra-chromosomal element, so that it is passed on to successivegenerations. In preferred embodiments, the PKSRP nucleic acid encodes aprotein comprising SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22,SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or SEQ ID NO:30.

The present invention also provides a method of modulating a plant'stolerance to an environmental stress comprising, modifying theexpression of a PKSRP coding nucleic acid in the plant. The plant'stolerance to the environmental stress can be increased or decreased asachieved by increasing or decreasing the expression of a PKSRP,respectively. Preferably, the plant's tolerance to the environmentalstress is increased by increasing expression of a PKSRP. Expression of aPKSRP can be modified by any method known to those of skill in the art.The methods of increasing expression of PKSRPs can be used wherein theplant is either transgenic or not transgenic. In cases when the plant istransgenic, the plant can be transformed with a vector containing any ofthe above described PKSRP coding nucleic acids, or the plant can betransformed with a promoter that directs expression of native PKSRP inthe plant, for example. The invention provides that such a promoter canbe tissue specific, developmentally regulated, or stress-inducible.Alternatively, non-transgenic plants can have native PKSRP expressionmodified by inducing a native promoter. The expression of PK-3 asdefined in SEQ ID NO:2, PK-4 as defined in SEQ ID NO:5, PK-10 as definedin SEQ ID NO:8, PK-11 as defined in SEQ ID NO:11, BnPK-1 as defined inSEQ ID NO:13, BnPK-2 as defined in SEQ ID NO:15, BnPK-3 as defined inSEQ ID NO:17, BnPK-4 as defined in SEQ ID NO:19, GmPK-1 as defined inSEQ ID NO:21, GmPK-2 as defined in SEQ ID NO:23, GmPK-3 as defined inSEQ ID NO:25, GmPK-4 as defined in SEQ ID NO:27, and OsPK-1 as definedin SEQ ID NO:29 in target plants can be accomplished by, but is notlimited to, one of the following examples: (a) constitutive promoter,(b) stress-inducible promoter, (c) chemical-induced promoter, and (d)engineered promoter overexpression with, for example, zinc-fingerderived transcription factors (Greisman and Pabo, 1997, Science275:657).

In a preferred embodiment, transcription of the PKSRP is modulated usingzinc-finger derived transcription factors (ZFPs) as described inGreisman and Pabo, 1997, Science 275:657 and manufactured by SangamoBiosciences, Inc. These ZFPs comprise both a DNA recognition domain anda functional domain that causes activation or repression of a targetnucleic acid such as a PKSRP nucleic acid. Therefore, activating andrepressing ZFPs can be created that specifically recognize the PKSRPpromoters described above and used to increase or decrease PKSRPexpression in a plant, thereby modulating the stress tolerance of theplant. The present invention also includes identification of thehomologs of SEQ ID NO:2, PK-4 as defined in SEQ ID NO:5, PK-10 asdefined in SEQ ID NO:8, PK-11 as defined in SEQ ID NO:11, BnPK-1 asdefined in SEQ ID NO:13, BnPK-2 as defined in SEQ ID NO:15, BnPK-3 asdefined in SEQ ID NO:17, BnPK-4 as defined in SEQ ID NO:19, GmPK-1 asdefined in SEQ ID NO:21, GmPK-2 as defined in SEQ ID NO:23, GmPK-3 asdefined in SEQ ID NO:25, GmPK-4 as defined in SEQ ID NO:27, and OsPK-1as defined in SEQ ID NO:29 in a target plant as well as the homolog'spromoter. The invention also provides a method of increasing expressionof a gene of interest within a host cell as compared to a wild typevariety of the host cell, wherein the gene of interest is transcribed inresponse to a PKSRP, comprising: (a) transforming the host cell with anexpression vector comprising a PKSRP coding nucleic acid, and (b)expressing the PKSRP within the host cell, thereby increasing theexpression of the gene transcribed in response to the PKSRP, as comparedto a wild type variety of the host cell.

In addition to introducing the PKSRP nucleic acid sequences intotransgenic plants, these sequences can also be used to identify anorganism as being Physcomitrella patens, Brassica napus, Glycine max,Oryza sativa, or a close relative thereof. Also, they may be used toidentify the presence of Physcomitrella patens, Brassica napus, Glycinemax, Oryza sativa, or a relative thereof in a mixed population ofmicroorganisms. The invention provides the nucleic acid sequences of anumber of Physcomitrella patens, Brassica napus, Glycine max, and Oryzasativa genes; by probing the extracted genomic DNA of a culture of aunique or mixed population of microorganisms under stringent conditionswith a probe spanning a region of a Physcomitrella patens, Brassicanapus, Glycine max, or Oryza sativa gene which is unique to thisorganism, one can ascertain whether this organism is present.

Further, the nucleic acid and polypeptide molecules of the invention mayserve as markers for specific regions of the genome. This has utilitynot only in the mapping of the genome, but also in functional studies ofPhyscomitrella patens, Brassica napus, Glycine max, and Oryza sativapolypeptides. For example, to identify the region of the genome to whicha particular Physcomitrella patens DNA-binding polypeptide binds, thePhyscomitrella patens genome could be digested, and the fragmentsincubated with the DNA-binding polypeptide. Those fragments that bindthe polypeptide may be additionally probed with the nucleic acidmolecules of the invention, preferably with readily detectable labels.Binding of such a nucleic acid molecule to the genome fragment enablesthe localization of the fragment to the genome map of Physcomitrellapatens, and, when performed multiple times with different enzymes,facilitates a rapid determination of the nucleic acid sequence to whichthe polypeptide binds. Further, the nucleic acid molecules of theinvention may be sufficiently identical to the sequences of relatedspecies such that these nucleic acid molecules may serve as markers forthe construction of a genomic map in related mosses.

The PKSRP nucleic acid molecules of the invention are also useful forevolutionary and polypeptide structural studies. The metabolic andtransport processes in which the molecules of the invention participateare utilized by a wide variety of prokaryotic and eukaryotic cells; bycomparing the sequences of the nucleic acid molecules of the presentinvention to those encoding similar enzymes from other organisms, theevolutionary relatedness of the organisms can be assessed. Similarly,such a comparison permits an assessment of which regions of the sequenceare conserved and which are not, which may aid in determining thoseregions of the polypeptide that are essential for the functioning of theenzyme. This type of determination is of value for polypeptideengineering studies and may give an indication of what the polypeptidecan tolerate in terms of mutagenesis without losing function.

Manipulation of the PKSRP nucleic acid molecules of the invention mayresult in the production of PKSRPs having functional differences fromthe wild-type PKSRPs. These polypeptides may be improved in efficiencyor activity, may be present in greater numbers in the cell than isusual, or may be decreased in efficiency or activity.

There are a number of mechanisms by which the alteration of a PKSRP ofthe invention may directly affect stress response and/or stresstolerance. In the case of plants expressing PKSRPs, increased transportcan lead to improved salt and/or solute partitioning within the planttissue and organs. By either increasing the number or the activity oftransporter molecules which exportionic molecules from the cell, it maybe possible to affect the salt tolerance of the cell.

The effect of the genetic modification in plants, C. glutamicum, fungi,algae, or ciliates on stress tolerance can be assessed by growing themodified microorganism or plant under less than suitable conditions andthen analyzing the growth characteristics and/or metabolism of theplant. Such analysis techniques are well known to one skilled in theart, and include dry weight, wet weight, polypeptide synthesis,carbohydrate synthesis, lipid synthesis, evapotranspiration rates,general plant and/or crop yield, flowering, reproduction, seed setting,root growth, respiration rates, photosynthesis rates, etc. (Applicationsof HPLC in Biochemistry in: Laboratory Techniques in Biochemistry andMolecular Biology, vol. 17; Rehm et al., 1993 Biotechnology, vol. 3,Chapter III: Product recovery and purification, page 469-714, VCH:Weinheim; Belter, P. A. et al., 1988, Bioseparations: downstreamprocessing for biotechnology, John Wiley and Sons; Kennedy, J. F. andCabral, J. M. S., 1992, Recovery processes for biological materials,John Wiley and Sons; Shaeiwitz, J. A. and Henry, J. D., 1988,Biochemical separations, in: Ulmann's Encyclopedia of IndustrialChemistry, vol. B3, Chapter 11, page 1-27, VCH: Weinheim; and Dechow, F.J., 1989, Separation and purification techniques in biotechnology, NoyesPublications).

For example, yeast expression vectors comprising the nucleic acidsdisclosed herein, or fragments thereof, can be constructed andtransformed into Saccharomyces cerevisiae using standard protocols. Theresulting transgenic cells can then be assayed for fail or alteration oftheir tolerance to drought, salt, and temperature stress. Similarly,plant expression vectors comprising the nucleic acids disclosed herein,or fragments thereof, can be constructed and transformed into anappropriate plant cell such as Arabidopsis, soy, rape, maize, wheat,Medicago truncatula, etc., using standard protocols. The resultingtransgenic cells and/or plants derived therefrom can then be assayed forfail or alteration of their tolerance to drought, salt, temperaturestress, and lodging.

The engineering of one or more PKSRP genes of the invention may alsoresult in PKSRPs having altered activities which indirectly impact thestress response and/or stress tolerance of algae, plants, ciliates, orfungi, or other microorganisms like C. glutamicum. For example, thenormal biochemical processes of metabolism result in the production of avariety of products (e.g., hydrogen peroxide and other reactive oxygenspecies) which may actively interfere with these same metabolicprocesses. For example, peroxynitrite is known to nitrate tyrosine sidechains, thereby inactivating some enzymes having tyrosine in the activesite (Groves, J. T., 1999, Curr. Opin. Chem. Biol. 3(2):226-235). Whilethese products are typically excreted, cells can be genetically alteredto transport more products than is typical for a wild-type cell. Byoptimizing the activity of one or more PKSRPs of the invention which areinvolved in the export of specific molecules, such as salt molecules, itmay be possible to improve the stress tolerance of the cell.

Additionally, the sequences disclosed herein, or fragments thereof, canbe used to generate knockout mutations in the genomes of variousorganisms, such as bacteria, mammalian cells, yeast cells, and plantcells (Girke, T., 1998, The Plant Journal 15:39-48). The resultantknockout cells can then be evaluated for their ability or capacity totolerate various stress conditions, their response to various stressconditions, and the effect on the phenotype and/or genotype of themutation. For other methods of gene inactivation, see U.S. Pat. No.6,004,804 “Non-Chimeric Mutational Vectors” and Puttaraju et al., 1999,Spliceosome-mediated RNA trans-splicing as a tool for gene therapy,Nature Biotechnology 17:246-252.

The aforementioned mutagenesis strategies for PKSRPs resulting inincreased stress resistance are not meant to be limiting; variations onthese strategies will be readily apparent to one skilled in the art.Using such strategies, and incorporating the mechanisms disclosedherein, the nucleic acid and polypeptide molecules of the invention maybe utilized to generate algae, ciliates, plants, fungi, or othermicroorganisms like C. glutamicum expressing mutated PKSRP nucleic acidand polypeptide molecules such that the stress tolerance is improved.

The present invention also provides antibodies that specifically bind toa PKSRP, or a portion thereof, as encoded by a nucleic acid describedherein. Antibodies can be made by many well-known methods (See, e.g.Harlow and Lane, “Antibodies; A Laboratory Manual,” Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., (1988)). Briefly, purified antigencan be injected into an animal in an amount and in intervals sufficientto elicit an immune response. Antibodies can either be purifieddirectly, or spleen cells can be obtained from the animal. The cells canthen fused with an immortal cell line and screened for antibodysecretion. The antibodies can be used to screen nucleic acid clonelibraries for cells secreting the antigen. Those positive clones canthen be sequenced. See, for example, Kelly et al., 1992, Bio/Technology10:163-167; Bebbington et al., 1992, Bio/Technology 10:169-175.

The phrases “selectively binds” and “specifically binds” with thepolypeptide refer to a binding reaction that is determinative of thepresence of the polypeptide in a heterogeneous population ofpolypeptides and other biologics. Thus, under designated immunoassayconditions, the specified antibodies bound to a particular polypeptidedo not bind in a significant amount to other polypeptides present in thesample. Selective binding of an antibody under such conditions mayrequire an antibody that is selected for its specificity for aparticular polypeptide. A variety of immunoassay formats may be used toselect antibodies that selectively bind with a particular polypeptide.For example, solid-phase ELISA immunoassays are routinely used to selectantibodies selectively immunoreactive with a polypeptide. See Harlow andLane, “Antibodies, A Laboratory Manual,” Cold Spring HarborPublications, New York, (1988), for a description of immunoassay formatsand conditions that could be used to determine selective binding.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious hosts. A description of techniques for preparing such monoclonalantibodies may be found in Stites et al., eds., “Basic and ClinicalImmunology,” (Lange Medical Publications, Los Altos, Calif., FourthEdition) and references cited therein, and in Harlow and Lane,“Antibodies, A Laboratory Manual,” Cold Spring Harbor Publications, NewYork, (1988).

Throughout this application, various publications are referenced. Thedisclosures of all of these publications and those references citedwithin those publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art to which this invention pertains.

It should also be understood that the foregoing relates to preferredembodiments of the present invention and that numerous changes may bemade therein without departing from the scope of the invention. Theinvention is further illustrated by the following examples, which arenot to be construed in any way as imposing limitations upon the scopethereof. On the contrary, it is to be clearly understood that resort maybe had to various other embodiments, modifications, and equivalentsthereof, which, after reading the description herein, may suggestthemselves to those skilled in the art without departing from the spiritof the present invention and/or the scope of the appended claims.

EXAMPLES Example 1

Growth of Physcomitrella patens Cultures

For this study, plants of the species Physcomitrella patens (Hedw.)B.S.G. from the collection of the genetic studies section of theUniversity of Hamburg were used. They originate from the strain 16/14collected by H. L. K. Whitehouse in Gransden Wood, Huntingdonshire(England), which was subcultured from a spore by Engel (1968, Am. J.Bot. 55, 438-446). Proliferation of the plants was carried out by meansof spores and by means of regeneration of the gametophytes. Theprotonema developed from the haploid spore as a chloroplast-richchloronema and chloroplast-low caulonema, on which buds formed afterapproximately 12 days. These grew to give gametophores bearingantheridia and archegonia. After fertilization, the diploid sporophytewith a short seta and the spore capsule resulted, in which themeiospores matured.

Culturing was carried out in a climatic chamber at an air temperature of25° C. and light intensity of 55 micromol s⁻¹m⁻² (white light; PhilipsTL 65 W/25 fluorescent tube) and a light/dark change of 16/8 hours. Themoss was either modified in liquid culture using Knop medium accordingto Reski and Abel (1985, Planta 165:354-358) or cultured on Knop solidmedium using 1% oxoid agar (Unipath, Basingstoke, England). Theprotonemas used for RNA and DNA isolation were cultured in aeratedliquid cultures. The protonemas were comminuted every 9 days andtransferred to fresh culture medium.

Example 2

Total DNA Isolation from Plants

The details for the isolation of total DNA relate to the working up ofone gram fresh weight of plant material. The materials used include thefollowing buffers: CTAB buffer: 2% (w/v)N-cethyl-N,N,N-trimethylammonium bromide (CTAB); 100 mM Tris HCl pH 8.0;1.4 M NaCl; 20 mM EDTA; N-Laurylsarcosine buffer: 10% (w/v)N-laurylsarcosine; 100 mM Tris HCl pH 8.0; 20 mM EDTA.

The plant material was triturated under liquid nitrogen in a mortar togive a fine powder and transferred to 2 ml Eppendorf vessels. The frozenplant material was then covered with a layer of 1 ml of decompositionbuffer (1 ml CTAB buffer, 100 μl of N-laurylsarcosine buffer, 20 μl ofβ-mercaptoethanol, and 10 μl of proteinase K solution, 10 mg/ml) andincubated at 60° C. for one hour with continuous shaking. The homogenateobtained was distributed into two Eppendorf vessels (2 ml) and extractedtwice by shaking with the same volume of chloroform/isoamyl alcohol(24:1). For phase separation, centrifugation was carried out at 8000×gand room temperature for 15 minutes in each case. The DNA was thenprecipitated at −70° C. for 30 minutes using ice-cold isopropanol. Theprecipitated DNA was sedimented at 4° C. and 10,000×g for 30 minutes andresuspended in 180 μl of TE buffer (Sambrook et al., 1989, Cold SpringHarbor Laboratory Press: ISBN 0-87969-309-6). For further purification,the DNA was treated with NaCl (1.2 M final concentration) andprecipitated again at −70° C. for 30 minutes using twice the volume ofabsolute ethanol. After a washing step with 70% ethanol, the DNA wasdried and subsequently taken up in 50 μl of H₂O+RNAse (50 mg/ml finalconcentration). The DNA was dissolved overnight at 4° C. and the RNAsedigestion was subsequently carried out at 37° C. for 1 hour. Storage ofthe DNA took place at 4° C.

Example 3

Isolation of Total RNA and poly-(A)+ RNA and cDNA Library Constructionfrom Physcomitrella patens

For the investigation of transcripts, both total RNA and poly-(A)⁺ RNAwere isolated. The total RNA was obtained from wild-type 9 day oldprotonemata following the GTC-method (Reski et al., 1994, Mol. Gen.Genet., 244:352-359). The Poly(A)+ RNA was isolated using Dyna Beads®(Dynal, Oslo, Norway) following the instructions of the manufacturer'sprotocol. After determination of the concentration of the RNA or of thepoly(A)+ RNA, the RNA was precipitated by addition of 1/10 volumes of 3M sodium acetate pH 4.6 and 2 volumes of ethanol and stored at −70° C.

For cDNA library construction, first strand synthesis was achieved usingMurine Leukemia Virus reverse transcriptase (Roche, Mannheim, Germany)and oligo-d(T)-primers, second strand synthesis by incubation with DNApolymerase I, Klenow enzyme and RNAseH digestion at 12° C. (2 hours),16° C. (1 hour), and 22° C. (1 hour). The reaction was stopped byincubation at 65° C. (10 minutes) and subsequently transferred to ice.Double stranded DNA molecules were blunted by T4-DNA-polymerase (Roche,Mannheim) at 37° C. (30 minutes). Nucleotides were removed byphenol/chloroform extraction and Sephadex G50 spin columns. EcoRIadapters (Pharmacia, Freiburg, Germany) were ligated to the cDNA ends byT4-DNA-ligase (Roche, 12° C., overnight) and phosphorylated byincubation with polynucleotide kinase (Roche, 37° C., 30 minutes). Thismixture was subjected to separation on a low melting agarose gel. DNAmolecules larger than 300 base pairs were eluted from the gel, phenolextracted, concentrated on Elutip-D-columns (Schleicher and Schuell,Dassel, Germany), and were ligated to vector arms and packed into lambdaZAPII phages or lambda ZAP-Express phages using the Gigapack Gold Kit(Stratagene, Amsterdam, Netherlands) using material and following theinstructions of the manufacturer.

Example 4

Sequencing and Function Annotation of Physcomitrella patens ESTs

cDNA libraries as described in Example 3 were used for DNA sequencingaccording to standard methods, and in particular, by the chaintermination method using the ABI PRISM Big Dye Terminator CycleSequencing Ready Reaction Kit (Perkin-Elmer, Weiterstadt, Germany).Random Sequencing was carried out subsequent to preparative plasmidrecovery from cDNA libraries via in vivo mass excision,retransformation, and subsequent plating of DH10B on agar plates(material and protocol details from Stratagene, Amsterdam, Netherlands).Plasmid DNA was prepared from overnight grown E. coli cultures grown inLuria-Broth medium containing ampicillin (See Sambrook et al., 1989,Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6) on a QiageneDNA preparation robot (Qiagen, Hilden) according to the manufacturer'sprotocols. Sequencing primers with the following nucleotide sequenceswere used:

5′-CAGGAAACAGCTATGACC-3′ SEQ ID NO:31 5′-CTAAAGGGAACAAAAGCTG-3′ SEQ IDNO:32 5′-TGTAAAACGACGGCCAGT-3′ SEQ ID NO:33

Sequences were processed and annotated using the software packageEST-MAX commercially provided by Bio-Max (Munich, Germany). The programincorporates practically all bioinformatics methods important forfunctional and structural characterization of polypeptide sequences. Themost important algorithms incorporated in EST-MAX are: FASTA (Verysensitive sequence database searches with estimates of statisticalsignificance; Pearson W. R., 1990, Rapid and sensitive sequencecomparison with FASTP and FASTA, Methods Enzymol. 183:63-98); BLAST(Very sensitive sequence database searches with estimates of statisticalsignificance; Altschul S. F. et al., Basic local alignment search tool,Journal of Molecular Biology 215:403-10); PREDATOR (High-accuracysecondary structure prediction from single and multiple sequences,Frishman, D. and Argos, P., 1997, 75% accuracy in polypeptide secondarystructure prediction, Polypeptides, 27:329-335); CLUSTALW (Multiplesequence alignment; Thompson, J. D. et al., 1994, CLUSTAL W: improvingthe sensitivity of progressive multiple sequence alignment throughsequence weighting, positions-specific gap penalties and weight matrixchoice. Nucleic Acids Research, 22:4673-4680); TMAP (Transmembraneregion prediction from multiply aligned sequences; Persson, B. andArgos, P., 1994, Prediction of transmembrane segments in polypeptidesutilizing multiple sequence alignments, J. Mol. Biol. 237:182-192);ALOM2 (Transmembrane region prediction from single sequences; Klein, P.et al., Prediction of polypeptide function from sequence properties: Adiscriminate analysis of a database. Biochim. Biophys. Acta 787:221-226(1984). Version 2 by Dr. K. Nakai); PROSEARCH (Detection of PROSITEpolypeptide sequence patterns; Kolakowski L. F. Jr. et al., 1992,ProSearch: fast searching of polypeptide sequences with regularexpression patterns related to polypeptide structure and function,Biotechniques 13:919-921); BLIMPS (Similarity searches against adatabase of ungapped blocks; J. C. Wallace and Henikoff S., 1992); andPATMAT (A searching and extraction program for sequence, pattern andblock queries and databases, CABIOS 8:249-254. Written by Bill Alford.).

Example 5

Identification of Physcomitrella patens ORFs Corresponding to PK-3,PK-4, PK-10, and PK-11

The Physcomitrella patens partial cDNAs (ESTs) for partial PK-3 (SEQ IDNO:1), partial PK-4 (SEQ ID NO:4), partial PK-10 (SEQ ID NO:7), andpartial PK-11 (SEQ ID NO:10) were identified in the Physcomitrellapatens EST sequencing program using the program EST-MAX through BLASTanalysis. These particular clones, which were found to encode ProteinKinases, were chosen for further analyses.

TABLE 1 Degree of Amino Acid Identity and Similarity of PK-3 and OtherKinases (Pairwise Comparison was used: gap penalty: 10; gap extensionpenalty: 0.1; score matrix: blosum62) Swiss-Prot # P51139 Q40518 P43288P43289 Q9LYJ6 Protein name Glycogen Shaggy- Shaggy- Shaggy- ProteinSynthase Related Related Related Kinase Kinase-3 Protein Protein ProteinMSK-3-Like Homolog MSK-3 Kinase NTK-1 Kinase Alpha Kinase Gamma SpeciesMedicago Nicotiana Arabidopsis Arabidopsis Arabidopsis sativa tabacumthaliana thaliana thaliana (Alfalfa) (Common (Mouse-ear (Mouse-ear(Mouse-ear tobacco) cress) cress) cress) Identity % 78% 79% 79% 80% 79%Similarity % 86% 86% 86% 87% 87%

TABLE 2 Degree of Amino Acid Identity and Similarity of PK-4 and OtherKinases (Pairwise Comparison was used: gap penalty: 10; gap extensionpenalty: 0.1; score matrix: blosum62) Swiss-Prot # Q9SZI1 Q9ZUP4 P42158Q39050 Q9LW62 Polypeptide COL-0 Putative Casein Casein Casein nameCasein Casein Kinase I, Kinase I Kinase Kinase I- Kinase I Delta LikeProtein Isoform Like Species Arabidopsis Arabidopsis ArabidopsisArabidopsis Arabidopsis thaliana thaliana thaliana thaliana thaliana(Mouse-ear (Mouse-ear (Mouse-ear (Mouse-ear (Mouse-ear cress) cress)cress) cress) cress) Identity % 35% 35% 37% 35% 35% Similarity % 46% 44%47% 45% 44%

TABLE 3 Degree of Amino Acid Identity and Similarity of PK-10 and aSimilar Protein (Pairwise Comparison was used: gap penalty: 10; gapextension penalty: 0.1; score matrix: blosum62) Sequence Gene PublicDatabase Sequence Similarity Name Sequence Protein Name Species Identity(%) (%) PK-10 AAG51974 Putative Leucine- Arabidopsis 45% 57% Rich Repeatthaliana Transmembrane Protein Kinase 1

TABLE 4 Degree of Amino Acid Identity and Similarity of PK-11 and aSimilar Protein (Pairwise Comparison was used: gap penalty: 10; gapextension penalty: 0.1; score matrix: blosum62) Sequence Gene PublicDatabase Sequence Similarity Name Sequence Protein Name Species Identity(%) (%) PK-11 AAK72257.1 CBL-Interacting Arabidopsis 64% 76% ProteinKinase thaliana 24

Example 6

Cloning of the Full-length Physcomitrella patens cDNA Encoding for PK-3,PK-4, PK-10, and PK-11

To isolate the clone encoding full-length PK-3 (SEQ ID NO:2), PCR wasperformed (as described below in Full-Length Amplification) using theoriginal ESTs described in Example 5 as template. The primers used foramplification are listed below in Table 5.

To isolate the clones encoding PK-4 (SEQ ID NO:5), PK-10 (SEQ ID NO:8),and PK-11 (SEQ ID NO:11) from Physcomitrella patens, cDNA libraries werecreated with SMART RACE cDNA Amplification kit (Clontech Laboratories)following the manufacturer's instructions. Total RNA isolated asdescribed in Example 3 was used as the template. The cultures weretreated prior to RNA isolation as follows: Salt Stress: 2, 6, 12, 24, 48hours with 1-M NaCl-supplemented medium; Cold Stress: 4° C. for the sametime points as for salt; Drought Stress: cultures were incubated on dryfilter paper for the same time points as for salt.

5′ RACE Protocol

The EST sequences of PK-4 (SEQ ID NO:4), PK-10 (SEQ ID NO:7), and PK-11(SEQ ID NO:10) identified from the database search as described inExample 5 were used to design oligos for RACE (See Table 5). Theextended sequence for these genes were obtained by performing RapidAmplification of cDNA Ends polymerase chain reaction (RACE PCR) usingthe Advantage 2 PCR kit (Clontech Laboratories) and the SMART RACE cDNAamplification kit (Clontech Laboratories) using a Biometra T3Thermocycler following the manufacturer's instructions. The sequencesobtained from the RACE reactions corresponded to full-length codingregion of PK-4, PK-10, and PK-11 and were used to design oligos forfull-length cloning of the respective gene (See below Full-LengthAmplification).

Full-length Amplification

A full-length clone corresponding to PK-3 (SEQ ID NO:2) was obtained byperforming polymerase chain reaction (PCR) with gene-specific primers(See Table 5) and the original EST as the template. The conditions forthe reaction were standard conditions with PWO DNA polymerase (Roche).PCR was performed according to standard conditions and to manufacturer'sprotocols (Sambrook et al., 1989, Molecular Cloning, A LaboratoryManual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., Biometra T3 Thermocycler). The parameters for the reactionwere: five minutes at 94° C. followed by five cycles of one minute at94° C., one minute at 50° C., and 1.5 minutes at 72° C. This wasfollowed by twenty five cycles of one minute at 94° C., one minute at65° C., and 1.5 minutes at 72° C.

Full-length clones for PK-4 (SEQ ID NO:5), PK-10 (SEQ ID NO:8), andPK-11 (SEQ ID NO:11) were isolated by repeating the RACE method butusing the gene-specific primers as given in Table 5.

The amplified fragments were extracted from agarose gel with a QIAquickGel Extraction Kit (Qiagen) and ligated into the TOPO pCR 2.1 vector(Invitrogen) following manufacturer's instructions. Recombinant vectorswere transformed into Top10 cells (Invitrogen) using standard conditions(Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual. 2ndEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).Transformed cells were selected for on LB agar containing 100 μg/mlcarbenicillin, 0.8 mg X-gal(5-bromo-4-chloro-3-indolyl-β-D-galactoside), and 0.8 mg IPTG(isopropylthio-β-D-galactoside) grown overnight at 37° C. White colonieswere selected and used to inoculate 3 ml of liquid LB containing 100μg/ml ampicillin and grown overnight at 37° C. Plasmid DNA was extractedusing the QIAprep Spin Miniprep Kit (Qiagen) following manufacturer'sinstructions. Analyses of subsequent clones and restriction mapping wasperformed according to standard molecular biology techniques (Sambrooket al., 1989, Molecular Cloning, A Laboratory Manual, 2nd Edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

TABLE 5 Scheme and Primers Used for Cloning of Full-Length Clones Sitesin the Isolation Primers Primers Gene final product Method Race RT-PCRPK-3 XmaI/SacI PCR of RC021: original 5′ATCCCGGGCGAG EST cloneTCTTCTATGGCATC TGCGACT3′ (SEQ ID NO:34) RC022: 5′ATGAGCTCAATATCAGGAGTTGCAC CCTTCAAC3′ (SEQ ID NO:35) PK-4 XmaI/EcoRV 5′ RACE andRC072: RC133N: RT-PCR for 5′TGTGTCTACGT 5′ATCCCGGGAGGC FL cloneGTCGCGGGGTCGA ATTGAACTACCTGG T3′ AGTGAG3′ (SEQ ID NO:36) (SEQ ID NO:37)RC134N: 5′GCGATATCGTTG AACTAGTAATCTGT GTTAACTT3′ (SEQ ID NO:38) PK-10XmaI/SacI 5′ RACE and NVT: RC580: RT-PCR for 5′CTGCGACGGA 5′ATCCCGGGTGTCFL clone AAACTCTCTTGC GGAATTCGGTCACA TGT3′ ATGAGCT3′ (SEQ ID NO:39)RC834: 5′GCGAGCTCGTGC GAATCATGTACTCC CATCACAC3′ (SEQ ID NO:40) PK-11XmaI/SacI 5′ RACE and RC253: RC1158: RT-PCR for 5′GCAGCGGTATA5′ATCCCGGGTTTC FL clone TCCTTGCTCCTCA TGGAATAGCTCAGA TC3′ AGCGT3′ RC520:RC1159: 5′CGATGTGAGAC 5′CGGAGCTCGATG GCCCTTGCTGTGG CAGCGGTATATCCT CA3′TGCTCCT3′ RC721: (SEQ ID NO:42) 5′GCAACGACTTG CCAGAACCTCGTG C3′ (SEQ IDNO:41)Tissue Harvest, RNA Isolation, and cDNA Library Construction

Canola, soybean, and rice plants were grown under a variety ofconditions and treatments, and different tissues were harvested atvarious developmental stages. Plant growth and harvesting were done in astrategic manner such that the probability of harvesting all expressablegenes in at least one or more of the resulting libraries is maximized.The mRNA was isolated as described in Example 3 from each of thecollected samples, and cDNA libraries were constructed. No amplificationsteps were used in the library production process in order to minimizeredundancy of genes within the sample and to retain expressioninformation. All libraries were 3′ generated from mRNA purified on oligodT columns. Colonies from the transformation of the cDNA library into E.coli were randomly picked and placed into microtiter plates.

Probe Hybridization

Plasmid DNA was isolated from the E. coli colonies and then spotted onmembranes. A battery of 288 ³³P radiolabeled 7-mer oligonucleotides weresequentially hybridized to these membranes. To increase throughput,duplicate membranes were processed. After each hybridization, a blotimage was captured during a phosphorimage scan to generate ahybridization profile for each oligonucleotide. This raw data image wasautomatically transferred via LIMS to a computer. Absolute identity wasmaintained by barcoding for the image cassette, filter, and orientationwithin the cassette. The filters were then treated using relatively mildconditions to strip the bound probes and returned to the hybridizationchambers for another round of hybridization. The hybridization andimaging cycle was repeated until the set of 288 oligomers was completed.

After completion of the hybridizations, a profile was generated for eachspot (representing a cDNA insert), as to which of the 288 ³³Pradiolabeled 7-mer oligonucleotides bound to that particular spot (cDNAinsert), and to what degree. This profile is defined as the signaturegenerated from that clone. Each clone's signature was compared with allother signatures generated from the same organism to identify clustersof related signatures. This process “sorts” all of the clones from anorganism into clusters before sequencing.

Gene Isolation

The clones were sorted into various clusters based on their havingidentical or similar hybridization signatures. A cluster should beindicative of the expression of an individual gene or gene family. Aby-product of this analysis is an expression profile for the abundanceof each gene in a particular library. One-path sequencing from the 5′end was used to predict the function of the particular clones bysimilarity and motif searches in sequence databases.

The full-length DNA sequence of the Physcomitrella patens PK-3 (SEQ IDNO:8) or PK-10 (SEQ ID NO:11) was blasted against proprietary contigdatabases of canola, rice, and soybean at E value of E-10. (Altschul,Stephen et al. Gapped BLAST and PSI_BLAST: a new generation of proteindatabase search program. Nucleic Acids Res. 25: 3389-3402). All thecontig hits were analyzed for the putative full length sequences, andthe longest clones representing the putative full length contigs werefully sequenced. Nine such contigs isolated from the proprietary contigdatabases are BnPK-1, BnPK-2, BnPK-3, BnPK-4, GmPK-1, GmPK-2, GmPK-3,GmPK-4, and OsPK-1. The homology of the BnPK-1, BnPK-2, BnPK-3, BnPK-4,GmPK-1, GmPK-2, GmPK-3, GmPK-4, and OsPK-1 amino acid sequences to theclosest prior art is indicated in Tables 6-14.

TABLE 6 Degree of Amino Acid Identity and Similarity of BnPK-1 and aSimilar Protein (Pairwise Comparison was used: gap penalty: 10; gapextension penalty: 0.1; score matrix: blosum62) Sequence Gene PublicDatabase Sequence Similarity Name Sequence Protein Name Species Identity(%) (%) BnPK-1 CAA55866 Shaggy/Glycogen Arabidopsis 93% 95% SynthaseKinase-3 thaliana Homologue

TABLE 7 Degree of Amino Acid Identity and Similarity of BnPK-2 and aSimilar Protein (Pairwise Comparison was used: gap penalty: 10; gapextension penalty: 0.1; score matrix: blosum62) Public Sequence GeneDatabase Protein Sequence Similarity Name Sequence Name Species Identity(%) (%) BnPK-2 CAB78873 Shaggy- Arabidopsis 98% 99% Like thalianaProtein Kinase Etha

TABLE 8 Degree of Amino Acid Identity and Similarity of BnPK-3 and aSimilar Protein (Pairwise Comparison was used: gap penalty: 10; gapextension penalty: 0.1; score matrix: blosum62) Public Sequence GeneDatabase Protein Sequence Similarity Name Sequence Name Species Identity(%) (%) BnPK-3 CAA11903 Shaggy- Arabidopsis 92% 94% Like thaliana KinaseBeta

TABLE 9 Degree of Amino Acid Identity and Similarity of BnPK-4 and aSimilar Protein (Pairwise Comparison was used: gap penalty: 10; gapextension penalty: 0.1; score matrix: blosum62) Sequence Gene PublicDatabase Sequence Similarity Name Sequence Protein Name Species Identity(%) (%) BnPK-4 AAG51974 Putative Leucine- Arabidopsis 87% 92% RichRepeat thaliana Transmembrane Protein Kinase 1

TABLE 10 Degree of Amino Acid Identity and Similarity of GmPK-1 and aSimilar Protein (Pairwise Comparison was used: gap penalty: 10; gapextension penalty: 0.1; score matrix: blosum62) Sequence Gene PublicDatabase Sequence Similarity Name Sequence Protein Name Species Identity(%) (%) GmPK-1 AAL36376 Putative Shaggy Arabidopsis 80% 87% ProteinKinase thaliana dzeta

TABLE 11 Degree of Amino Acid Identity and Similarity of GmPK-2 and aSimilar Protein (Pairwise Comparison was used: gap penalty: 10; gapextension penalty: 0.1; score matrix: blosum62) Public Sequence GeneDatabase Sequence Similarity Name Sequence Protein Name Species Identity(%) (%) GmPK-2 AAG50665 Putative Glycogen Arabidopsis 85% 92% SynthaseKinase thaliana

TABLE 12 Degree of Amino Acid Identity and Similarity of GmPK-3 and aSimilar Protein (Pairwise Comparison was used: gap penalty: 10; gapextension penalty: 0.1; score matrix: blosum62) Public Sequence SequenceGene Database Protein Identity Similarity Name Sequence Name Species (%)(%) GmPK-3 AAK93730 Putative Arabidopsis 85% 89% Shaggy thaliana Kinase

TABLE 13 Degree of Amino Acid Identity and Similarity of GmPK-4 and aSimilar Protein (Pairwise Comparison was used: gap penalty: 10; gapextension penalty: 0.1; score matrix: blosum62) Sequence Gene PublicDatabase Sequence Similarity Name Sequence Protein Name Species Identity(%) (%) GmPK-4 AAL59961 Putative Leucine- Arabidopsis 58% 68% RichRepeat thaliana Transmembrane Protein Kinase

TABLE 14 Degree of Amino Acid Identity and Similarity of OsPK-1 and aSimilar Protein (Pairwise Comparison was used: gap penalty: 10; gapextension penalty: 0.1; score matrix: blosum62) Public Sequence GeneDatabase Protein Sequence Similarity Name Sequence Name Species Identity(%) (%) OsPK-1 CAA48474 Protein Medicago 89% 90% Kinase sativa

Example 7

Engineering Stress-tolerant Arabidopsis Plants by Over-expressing theGenes PK-3 and PK-4

Binary Vector Construction: pBPS-JH001

The plasmid construct pLMNC53 (Mankin, 2000, Ph.D. thesis, University ofNorth Carolina) was digested with HindIII (Roche) and blunt-end filledwith Klenow enzyme and 0.1 mM dNTPs according to manufacturer'sinstructions. This fragment was purified by agarose gel and extractedvia the QIAquick Gel Extraction kit (Qiagen) according to manufacturer'sinstructions. The purified fragment was then digested with EcoRI(Roche), purified by agarose gel, and extracted via the QIAquick GelExtraction kit (Qiagen) according to manufacturer's instructions. Theresulting 1.4 kilobase fragment, the gentamycin cassette, included thenos promoter, aacCI gene, and the g7 terminator.

The vector pBlueScript was digested with EcoRI and SmaI (Roche)according to manufacturer's instructions, and the resulting fragment wasextracted from agarose gel with a QIAquick Gel Extraction Kit (Qiagen)according to manufacturer's instructions. The digested pBlueScriptvector and the gentamycin cassette fragments were ligated with T4 DNALigase (Roche) according to manufacturer's instructions, joining the tworespective EcoRI sites and joining the blunt-ended HindIII site with theSmaI site.

The recombinant vector (pGMBS) was transformed into Top10 cells(Invitrogen) using standard conditions. Transformed cells were selectedfor on LB agar containing 100 μg/ml carbenicillin, 0.8 mg X-gal(5-bromo-4-chloro-3-indolyl-β-D-galactoside) and 0.8 mg IPTG(isopropylthio-β-D-galactoside), grown overnight at 37° C. Whitecolonies were selected and used to inoculate 3 ml of liquid LBcontaining 100 μg/ml ampicillin and grown overnight at 37° C. PlasmidDNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen) followingmanufacturer's instructions. Analyses of subsequent clones andrestriction mapping were performed according to standard molecularbiology techniques (Sambrook et al., 1989, Molecular Cloning, ALaboratory Manual. 2nd Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.).

Both the pGMBS vector and p1bxSuperGUS vector were digested with XbaIand KpnI (Roche) according to manufacturer's instructions, excising thegentamycin cassette from pGMBS and producing the backbone from thep1bxSuperGUS vector. The resulting fragments were extracted from agarosegel with a QIAquick Gel Extraction Kit (Qiagen) according tomanufacturer's instructions. These two fragments were ligated with T4DNA ligase (Roche) according to manufacturer's instructions.

The resulting recombinant vector (pBPS-JH001) was transformed into Top10cells (Invitrogen) using standard conditions. Transformed cells wereselected for on LB agar containing 100 μg/ml carbenicillin, 0.8 mg X-gal(5-bromo-4-chloro-3-indolyl-β-D-galactoside) and 0.8 mg IPTG(isopropylthio-β-D-galactoside), grown overnight at 37° C. Whitecolonies were selected and used to inoculate 3 ml of liquid LBcontaining 100 μg/ml ampicillin and grown overnight at 37° C. PlasmidDNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen) followingmanufacturer's instructions. Analyses of subsequent clones andrestriction mapping were performed according to standard molecularbiology techniques (Sambrook et al., 1989, Molecular Cloning, ALaboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.).

Binary Vector Construction: pBPS-SC022

The plasmid construct pACGH101 was digested with PstI (Roche) and FseI(NEB) according to manufacturers' instructions. The fragment waspurified by agarose gel and extracted via the Qiaex II DNA Extractionkit (Qiagen). This resulted in a vector fragment with the ArabidopsisActin2 promoter with internal intron and the OCS3 terminator.

Primers for PCR amplification of the NPTII gene were designed[5′NPT-Pst: GCG-CTG-CAG-ATT-TCA-TTT-GGA-GAG-GAC-ACG (SEQ ID NO:39);3′NPT-Fse: CGC-GGC-CGG-CCT-CAG-AAG-AAC-TCG-TCA-AGA-AGG-CG (SEQ IDNO:40)]. The 0.9 kilobase NPTII gene was amplified via PCR from pCambia2301 plasmid DNA using the following parameters: 94° C. 60 sec, {94° C.60 sec, 61° C. (−0.1° C. per cycle) 60 sec, 72° C. 2 min}×25 cycles, 72°C. 10 min on Biometra T-Gradient machine. The amplified product waspurified via the Qiaquick PCR Extraction kit (Qiagen) followingmanufacturer's instructions. The PCR DNA was then subcloned into thepCR-BluntII TOPO vector (Invitrogen) following manufacturer'sinstructions (NPT-Topo construct). These ligations were transformed intoTop10 cells (Invitrogen) and grown on LB plates with 50 μg/ml kanamycinsulfate overnight at 37° C. Colonies were then used to inoculate 2 ml LBmedia with 50 μg/ml kanamycin sulfate and grown overnight at 37° C.Plasmid DNA was recovered using the Qiaprep Spin Miniprep kit (Qiagen)and sequenced in both the 5′ and 3′ directions using standardconditions. Subsequent analysis of the sequence data using Vector NTIsoftware revealed that there were not any PCR errors introduced in theNPTII gene sequence.

The NPT-Topo construct was then digested with PstI (Roche) and FseI(NEB) according to manufacturers' instructions. The 0.9 kilobasefragment was purified on agarose gel and extracted by Qiaex II DNAExtraction kit (Qiagen). The Pst/Fse insert fragment from NPT-Topo andthe Pst/Fse vector fragment from pACGH101 were then ligated togetherusing T4 DNA Ligase (Roche) following manufacturer's instructions. Theligation reaction was then transformed into Top10 cells (Invitrogen)under standard conditions, creating pBPS-sc019 construct. Colonies wereselected on LB plates with 50 μg/ml kanamycin sulfate and grownovernight at 37° C. These colonies were then used to inoculate 2 ml LBmedia with 50 μg/ml kanamycin sulfate and grown overnight at 37° C.Plasmid DNA was recovered using the Qiaprep Spin Miniprep kit (Qiagen)following the manufacturer's instructions.

The pBPS-SC019 construct was digested with KpnI and BsaI (Roche)according to manufacturer's instructions. The fragment was purified viaagarose gel and then extracted via the Qiaex II DNA Extraction kit(Qiagen) as per its instructions, resulting in a 3 kilobase Act-NPTcassette, which included the Arabidopsis Actin2 promoter with internalintron, the NPTII gene, and the OCS3 terminator.

The pBPS-JH001 vector was digested with SpeI and ApaI (Roche) andblunt-end filled with Klenow enzyme and 0.1 mM dNTPs (Roche) accordingto manufacturer's instructions. This produced a 10.1 kilobase vectorfragment minus the Gentamycin cassette, which was recircularized byself-ligating with T4 DNA Ligase (Roche), and transformed into Top10cells (Invitrogen) via standard conditions. Transformed cells wereselected for on LB agar containing 50 μg/ml kanmycin sulfate and grownovernight at 37° C. Colonies were then used to inoculate 2 ml of liquidLB containing 50 μg/ml kanamycin sulfate and grown overnight at 37° C.Plasmid DNA was extracted using the QIAprep Spin Miniprep Kit (Qiagen)following manufacturer's instructions. The recircularized plasmid wasthen digested with KpnI (Roche) and extracted from agarose gel via theQiaex II DNA Extraction kit (Qiagen) according to manufacturers'instructions.

The Act-NPT Kpn-cut insert and the Kpn-cut pBPS-JH001 recircularizedvector were then ligated together using T4 DNA Ligase (Roche) andtransformed into Top10 cells (Invitrogen) according to manufacturers'instructions. The resulting construct, pBPS-SC022, now contained theSuper Promoter, the GUS gene, the NOS terminator, and the Act-NPTcassette. Transformed cells were selected for on LB agar containing 50μg/ml kanmycin sulfate and grown overnight at 37° C. Colonies were thenused to inoculate 2 ml of liquid LB containing 50 μg/ml kanamycinsulfate and grown overnight at 37° C. Plasmid DNA was extracted usingthe QIAprep Spin Miniprep Kit (Qiagen) following manufacturer'sinstructions. After confirmation of ligation success via restrictiondigests, pBPS-sc022 plasmid DNA was further propagated and recoveredusing the Plasmid Midiprep Kit (Qiagen) following the manufacturer'sinstructions.

Analyses of clones by restriction mapping was performed according tostandard molecular biology techniques (Sambrook et al., 1989, MolecularCloning, A Laboratory Manual, 2nd Edition, Cold Spring HarborLaboratory).

Subcloning of PK-3, PK-4 PK-10, and PK-11 into the Binary Vectors

The fragments containing the different Physcomitrella patens polypeptidekinases were subcloned from the recombinant PCR2.1 TOPO vectors bydouble digestion with restriction enzymes (See Table 15) according tomanufacturer's instructions. The subsequent fragment was excised fromagarose gel with a QIAquick Gel Extraction Kit (Qiagen) according tomanufacturer's instructions and ligated into the binary vectorpBPS-JH001 or pBPS-SC022 which was cleaved with appropriate enzymes (SeeTable 15) and dephosphorylated prior to ligation. The resultingrecombinant vectors (See Table 15) contained the correspondingPolypeptide Kinase in the sense orientation under the constitutive superpromoter.

TABLE 15 Listed are the names of the various constructs of thePhyscomitrella patens Polypeptide Kinases used for plant transformationEnzymes Used Enzymes Used to Restrict Binary to Generate the BinaryBinary Vector Gene Vector Gene Fragment Vector Construct PK-3 pBPS-JH001XmaI/SacI XmaI/SacI pBPS-LVM071 PK-4 pBPS-JH001 XmaI/EcoRV XmaI/Ecl136pBPS-LVM015 PK-10 pBPS-SC022 XmaI/SacI XmaI/SacI pBPS-ERG015 PK-11pBPS-SC022 XmaI/SacI XmaI/SacI pBPS-LVM230Agrobacterium Transformation

The recombinant vectors were transformed into Agrobacterium tumefaciensC58C1 and PMP90 according to standard conditions (Hoefgen andWillmitzer, 1990).

Plant Transformation

Arabidopsis thaliana ecotype C24 were grown and transformed according tostandard conditions (Bechtold, 1993, Acad. Sci. Paris. 316:1194-1199;Bent et al., 1994, Science 265:1856-1860).

Screening of Transformed Plants

T1 seeds were sterilized according to standard protocols (Xiong et al.,1999, Plant Molecular Biology Reporter 17: 159-170). Seeds were platedon ½ Murashige and Skoog media (MS) pH 5.7 with KOH (Sigma-Aldrich),0.6% agar and supplemented with 1% sucrose, 2 μg/ml benomyl(Sigma-Aldrich), and 150 μg/ml gentamycin (Sigma-Aldrich) (pBPS-JH001transformants) or 50 μg/ml kanamycin (pBPS-SC022 transformants). Seedson plates were vernalized for four days at 4° C. The seeds weregerminated in a climatic chamber at an air temperature of 22° C. andlight intensity of 40 micromol s⁻¹m⁻² (white light; Philips TL 65 W/25fluorescent tube) and 16 hours light and 8 hours dark day length cycle.Transformed seedlings were selected after 14 days and transferred to ½MS media pH 5.7 with KOH 0.6% agar plates supplemented with 1% sucrose,0.5 g/L MES (Sigma-Aldrich), and 2 μg/ml benomyl (Sigma-Aldrich) andallowed to recover for five to seven days.

Drought Tolerance Screening

T1 seedlings were transferred to dry, sterile filter paper in a petridish and allowed to desiccate for two hours at 80% RH (relativehumidity) in a Sanyo Growth Cabinet MLR-350H, micromole s⁻¹m⁻² (whitelight; Philips TL 65 W/25 fluorescent tube). The RH was then decreasedto 60%, and the seedlings were desiccated further for eight hours.Seedlings were then removed and placed on ½ MS 0.6% agar platessupplemented with 2 μg/ml benomyl (Sigma-Aldrich) and 0.5 g/L MES(Sigma-Aldrich) and scored after five days.

Under drought stress conditions, PK-3 over-expressing Arabidopsisthaliana plants showed a 54% survival rate (7 survivors from 13 stressedplants) to the stress screening, whereas the untransformed control onlyshowed a 6% survival rate (1 survivor from 18 stressed plants). It isnoteworthy that the analyses of these transgenic lines were performedwith T1 plants, and therefore, the results will be better when ahomozygous, strong expresser is found.

Transgenic plants overexpressing the PKSRP are screened for theirimproved drought tolerance, demonstrating that transgene expressionconfers drought tolerance.

TABLE 16 Summary of the drought stress tests Drought Stress Test Totalnumber of Percentage of Gene Name Number of survivors plants survivorsPK-3 7 13 54% Control 1 18  6%“In-Soil” Drought Tolerance Screening

T1 seeds were sterilized in 100% bleach, 0.01% TritonX for five minutestwo times and rinsed five times with sterile ddH2O. The sterile seedswere plated onto selection plates (½ MS, 0.6% phytagar, 0.5 g/L MES, 1%sucrose, 2 μg/ml benamyl, 50 μg/ml kanamycin, 0.6% agar). Plates wereincubated at 4° C. for 4 days in the dark.

Plates were then moved for to 22° C. under continuous light for 10 daysfor germination and concomitant selection for transgenic plants.Seedlings were transplanted at the 4-5-leaf stage into 5.5 cm diameterpots filled with loosely packed soil (Metromix 360, Scotts) wetted with1 g/L 20-20-20 fertilizer (Peters Professional, Scotts). Pots wereplaced randomly on trays with 5 control plants (transformed lines withempty vector) in each tray. Trays were placed randomly in the growthchamber.

Plants were grown (22° C., continuous light) for approximately sevendays, watering as needed. Watering was stopped at the time when themajority of the plants was about to bolt, and this point was denoted day“0” of the assay. After this day, trays were turned 180° every other dayto minimize local drying patterns. The assay was stopped approximatelyat day 12-19, depending on the speed of drying of the pots containingthe controls. Pots were then watered and survival rates were determinedafter 5 days.

PK-10 overexpressing Arabidopsis thaliana plants showed a 60% survivalrate (6 survivors from 10 stressed plants) to the stress screening.PK-11 over-expressing Arabidopsis thaliana plants showed a 65% survivalrate (11 survivors from 17 stressed plants) to the stress screening.This survival rate is significantly higher, 99% confidence interval,than that of the control. It is noteworthy that these analyses wereperformed with T1 plants. The results should be better when ahomozygous, strong expresser is found.

TABLE 17 Summary of the drought stress tests Drought Test Summay Numberof Total Number Percentage of Gene Name survivors of plants survivorsPpPK-10 6 10 60% PpPK-11 11 17 65%HS=significant difference with 99% confidence interval on a z-testFreezing Tolerance Screening

Seedlings are moved to petri dishes containing ½ MS 0.6% agarsupplemented with 2% sucrose and 2 μg/ml benomyl. After four days, theseedlings are incubated at 4° C. for 1 hour and then covered with shavedice. The seedlings are then placed in an Environmental Specialist ES2000Environmental Chamber and incubated for 3.5 hours beginning at −1.0° C.,decreasing 1° C./hour. The seedlings are then incubated at −5.0° C. for24 hours and then allowed to thaw at 5° C. for 12 hours. The water ispoured off and the seedlings are scored after 5 days. Transgenic plantsover-expressing PK-3 and PK-4 are screened for their improved freezingtolerance demonstrating that transgene expression confers freezingtolerance.

Salt Tolerance Screening

Seedlings are transferred to filter paper soaked in ½ MS and placed on ½MS 0.6% agar supplemented with 2 μg/ml benomyl the night before the salttolerance screening. For the salt tolerance screening, the filter paperwith the seedlings is moved to stacks of sterile filter paper, soaked in50 mM NaCl, in a petri dish. After two hours, the filter paper with theseedlings is moved to stacks of sterile filter paper, soaked with 200 mMNaCl, in a petri dish. After two hours, the filter paper with theseedlings is moved to stacks of sterile filter paper, soaked in 600 mMNaCl, in a petri dish. After 10 hours, the seedlings are moved to petridishes containing ½ MS 0.6% agar supplemented with 2 μg/ml benomyl. Theseedlings are scored after 5 days. The transgenic plants are screenedfor their improved salt tolerance demonstrating that transgeneexpression confers salt tolerance.

Example 8

Detection of the PK-3 and PK-4 Transgenes in the Transgenic ArabidopsisLines

To check for the presence of the PK-3 and PK-4 transgenes in transgenicArabidopsis lines, PCR was performed on genomic DNA which contaminatesthe RNA samples taken as described in Example 9 below. Two and one halfmicroliters of the RNA sample was used in a 50 μl PCR reaction using TaqDNA polymerase (Roche Molecular Biochemicals) according to themanufacturer's instructions.

Binary vector plasmid with each gene cloned in was used as positivecontrol, and the wild-type C24 genomic DNA was used as negative controlin the PCR reactions. Ten μl of the PCR reaction was analyzed on 0.8%agarose-ethidium bromide gel.

PK-3: The primers used in the reactions were:

5′CGAGAGCTGCAGATCATGCGACTGTTG3′ (SEQ ID NO:41)5′GCTCTGCCATCACGCAACCCATCGAC 3′ (SEQ ID NO:42)

The PCR program was as following: 35 cycles of 1 minute at 94° C., 30seconds at 62° C., and 1 minute at 72° C., followed by 5 minutes at 72°C. A 0.45 kilobase fragment was produced from the positive control andthe transgenic plants.

PK-4: The primers used in the reactions were:

(SEQ ID NO:37) 5′ATCCCGGGAGGCATTGAACTACCTGGAGTGAG3′ (SEQ ID NO:43)5′GCGATATCGTTGAACTAGTAATCTGTGTTAACTTTATC3′

The PCR program was as following: 30 cycles of 1 minute at 94° C., 1minute at 62° C., and 4 minutes at 72° C., followed by 10 minutes at 72°C. A 1.7 kilobase fragment was produced from the positive control andthe transgenic plants.

The transgenes were successfully amplified from the T1 transgenic lines,but not from the wild type C24. This result indicates that the T1transgenic plants contain at least one copy of the transgenes. There wasno indication of existence of either identical or very similar genes inthe untransformed Arabidopsis thaliana control which could be amplifiedby this method from the wild-type plants.

Example 9

Detection of the PK-3 and PK-4 Transgene mRNA in Transgenic ArabidopsisLines

Transgene expression was detected using RT-PCR. Total RNA was isolatedfrom stress-treated plants using a procedure adapted from Verwoerd etal., 1989, NAR 17:2362). Leaf samples (50-100 mg) were collected andground to a fine powder in liquid nitrogen. Ground tissue wasresuspended in 500 μl of a 80° C., 1:1 mixture, of phenol to extractionbuffer (100 mM LiCl, 100 mM Tris pH8, 10 mM EDTA, 1% SDS), followed bybrief vortexing to mix. After the addition of 250 μl of chloroform, eachsample was vortexed briefly. Samples were then centrifuged for 5 minutesat 12,000×g. The upper aqueous phase was removed to a fresh eppendorftube. RNA was precipitated by adding 1/10^(th) volume 3 M sodium acetateand 2 volumes 95% ethanol. Samples were mixed by inversion and placed onice for 30 minutes. RNA was pelleted by centrifugation at 12,000×g for10 minutes. The supernatant was removed and pellets briefly air-dried.RNA sample pellets were resuspended in 10 μl DEPC treated water. Toremove contaminating DNA from the samples, each was treated withRNase-free DNase (Roche) according to the manufacturer'srecommendations. cDNA was synthesized from total RNA using theSuperscript First-Strand Synthesis System for RT-PCR (Gibco-BRL)following manufacturer's recommendations.

PCR amplification of a gene-specific fragment from the synthesized cDNAwas performed using Taq DNA polymerase (Roche) and gene-specific primersdescribed in Example 8 in the following reaction: 1×PCR buffer, 1.5 mMMgCl₂, 0.2 μM each primer, 0.2 μM dNTPs, 1 unit polymerase, 5 μl cDNAfrom synthesis reaction. Amplification was performed under the followingconditions: denaturation, 95° C., 1 minute; annealing, 62° C., 30seconds; extension, 72° C., 1 minute, 35 cycles; extension, 72° C., 5minutes; hold, 4° C., forever. PCR products were run on a 1% agarosegel, stained with ethidium bromide, and visualized under UV light usingthe Quantity-One gel documentation system (Bio-Rad).

Expression of the transgenes was detected in the T1 transgenic line.This result indicated that the transgenes are expressed in thetransgenic lines and suggested that their gene product improved plantstress tolerance in the transgenic line. In agreement with the previousstatement, no expression of identical or very similar endogenous genescould be detected by this method. These results are in agreement withthe data from Example 8.

Example 10

Engineering Stress-tolerant Soybean Plants by Over-expressing the PK-3,PK-4, PK-10, and PK-11 Genes

The constructs pBPS-LVM071, pBPS-LVM015, pBPS-ERG015, and pBPS-LVM230are used to transform soybean as described below.

Seeds of soybean are surface sterilized with 70% ethanol for 4 minutesat room temperature with continuous shaking, followed by 20% (v/v)Clorox supplemented with 0.05% (v/v) Tween for 20 minutes withcontinuous shaking. Then, the seeds are rinsed 4 times with distilledwater and placed on moistened sterile filter paper in a Petri dish atroom temperature for 6 to 39 hours. The seed coats are peeled off, andcotyledons are detached from the embryo axis. The embryo axis isexamined to make sure that the meristematic region is not damaged. Theexcised embryo axes are collected in a half-open sterile Petri dish andair-dried to a moisture content less than 20% (fresh weight) in a sealedPetri dish until further use.

Agrobacterium tumefaciens culture is prepared from a single colony in LBsolid medium plus appropriate antibiotics (e.g. 100 mg/l streptomycin,50 mg/l kanamycin) followed by growth of the single colony in liquid LBmedium to an optical density at 600 nm of 0.8. Then, the bacteriaculture is pelleted at 7000 rpm for 7 minutes at room temperature, andresuspended in MS (Murashige and Skoog, 1962) medium supplemented with100 μM acetosyringone. Bacteria cultures are incubated in thispre-induction medium for 2 hours at room temperature before use. Theaxis of soybean zygotic seed embryos at approximately 15% moisturecontent are imbibed for 2 hours at room temperature with the pre-inducedAgrobacterium suspension culture. The embryos are removed from theimbibition culture and transferred to Petri dishes containing solid MSmedium supplemented with 2% sucrose and incubated for 2 days, in thedark at room temperature.

Alternatively, the embryos are placed on top of moistened (liquid MSmedium) sterile filter paper in a Petri dish and incubated under thesame conditions described above. After this period, the embryos aretransferred to either solid or liquid MS medium supplemented with 500mg/L carbenicillin or 300 mg/L cefotaxime to kill the agrobacteria. Theliquid medium is used to moisten the sterile filter paper. The embryosare incubated during 4 weeks at 25° C., under 150 μmol m⁻²sec⁻¹ and 12hours photoperiod. Once the seedlings have produced roots, they aretransferred to sterile metromix soil. The medium of the in vitro plantsis washed off before transferring the plants to soil. The plants arekept under a plastic cover for 1 week to favor the acclimatizationprocess. Then the plants are transferred to a growth room where they areincubated at 25° C., under 150 μmol m⁻² sec⁻¹ light intensity and 12hours photoperiod for about 80 days.

The transgenic plants are then screened for their improved drought,salt, and/or cold tolerance according to the screening method describedin Example 7, demonstrating that transgene expression confers stresstolerance.

Example 11

Engineering Stress-tolerant Rapeseed/Canola Plants by Over-expressingthe PK-3, PK-4, PK-10, and PK-11 Genes

The constructs pBPS-LVM071, pBPS-LVM015, pBPS-ERG015, and pBPS-LVM230are used to transform rapeseed/canola as described below.

The method of plant transformation described herein is applicable toBrassica and other crops. Seeds of canola are surface sterilized with70% ethanol for 4 minutes at room temperature with continuous shaking,followed by 20% (v/v) Clorox supplemented with 0.05% (v/v) Tween for 20minutes, at room temperature with continuous shaking. Then, the seedsare rinsed 4 times with distilled water and placed on moistened sterilefilter paper in a Petri dish at room temperature for 18 hours. Then theseed coats are removed, and the seeds are air dried overnight in ahalf-open sterile Petri dish. During this period, the seeds loseapproximately 85% of its water content. The seeds are then stored atroom temperature in a sealed Petri dish until further use. DNAconstructs and embryo imbibition are as described in Example 10. Samplesof the primary transgenic plants (T0) are analyzed by PCR to confirm thepresence of T-DNA. These results are confirmed by Southern hybridizationin which DNA is electrophoresed on a 1% agarose gel and transferred to apositively charged nylon membrane (Roche Diagnostics). The PCR DIG ProbeSynthesis Kit (Roche Diagnostics) is used to prepare adigoxigenin-labelled probe by PCR, and used as recommended by themanufacturer.

The transgenic plants are then screened for their improved stresstolerance according to the screening method described in Example 7,demonstrating that transgene expression confers stress tolerance.

Example 12

Engineering Stress-tolerant Corn Plants by Over-expressing the PK-3,PK-4, PK-10, and PK-11 Genes

The constructs pBPS-LVM071, pBPS-LVM015, pBPS-ERG015, and pBPS-LVM230are used to transform corn as described below.

Transformation of maize (Zea Mays L.) is performed with the methoddescribed by Ishida et al., 1996, Nature Biotech. 14745-50. Immatureembryos are co-cultivated with Agrobacterium tumefaciens that carry“super binary” vectors, and transgenic plants are recovered throughorganogenesis. This procedure provides a transformation efficiency ofbetween 2.5% and 20%. The transgenic plants are then screened for theirimproved drought, salt, and/or cold tolerance according to the screeningmethod described in Example 7, demonstrating that transgene expressionconfers stress tolerance.

Example 13

Engineering Stress-tolerant Wheat Plants by Over-expressing the PK-3,PK-4, PK-10, and PK-11 Genes

The constructs pBPS-LVM071, pBPS-LVM015, pBPS-ERG015, and pBPS-LVM230are used to transform wheat as described below.

Transformation of wheat is performed with the method described by Ishidaet al., 1996, Nature Biotech. 14745-50. Immature embryos areco-cultivated with Agrobacterium tumefaciens that carry “super binary”vectors, and transgenic plants are recovered through organogenesis. Thisprocedure provides a transformation efficiency between 2.5% and 20%. Thetransgenic plants are then screened for their improved stress toleranceaccording to the screening method described in Example 7, demonstratingthat transgene expression confers stress tolerance.

Example 14

Identification of Identical and Heterologous Genes

Gene sequences can be used to identify identical or heterologous genesfrom cDNA or genomic libraries. Identical genes (e.g. full-length cDNAclones) can be isolated via nucleic acid hybridization using for examplecDNA libraries. Depending on the abundance of the gene of interest,100,000 up to 1,000,000 recombinant bacteriophages are plated andtransferred to nylon membranes. After denaturation with alkali, DNA isimmobilized on the membrane by e.g. UV cross linking. Hybridization iscarried out at high stringency conditions. In aqueous solution,hybridization and washing is performed at an ionic strength of 1 M NaCland a temperature of 68° C. Hybridization probes are generated by e.g.radioactive (³²P) nick transcription labeling (High Prime, Roche,Mannheim, Germany). Signals are detected by autoradiography.

Partially identical or heterologous genes that are related but notidentical can be identified in a manner analogous to the above-describedprocedure using low stringency hybridization and washing conditions. Foraqueous hybridization, the ionic strength is normally kept at 1 M NaClwhile the temperature is progressively lowered from 68 to 42° C.

Isolation of gene sequences with homology (or sequenceidentity/similarity) only in a distinct domain of (for example 10-20amino acids) can be carried out by using synthetic radio labeledoligonucleotide probes. Radiolabeled oligonucleotides are prepared byphosphorylation of the 5-prime end of two complementary oligonucleotideswith T4 polynucleotide kinase. The complementary oligonucleotides areannealed and ligated to form concatemers. The double strandedconcatemers are than radiolabeled by, for example, nick transcription.Hybridization is normally performed at low stringency conditions usinghigh oligonucleotide concentrations.

Oligonucleotide Hybridization Solution:

-   6×SSC-   0.01 M sodium phosphate-   1 mM EDTA (pH 8)-   0.5% SDS-   100 μg/ml denatured salmon sperm DNA-   0.1% nonfat dried milk

During hybridization, temperature is lowered stepwise to 5-10° C. belowthe estimated oligonucleotide T_(m) or down to room temperature followedby washing steps and autoradiography. Washing is performed with lowstringency such as 3 washing steps using 4×SSC. Further details aredescribed by Sambrook, J. et al., 1989, “Molecular Cloning: A LaboratoryManual,” Cold Spring Harbor Laboratory Press or Ausubel, F. M. et al.,1994, “Current Protocols in Molecular Biology,” John Wiley & Sons.

Example 15

Identification of Identical Genes by Screening Expression Libraries withAntibodies

cDNA clones can be used to produce recombinant polypeptide for examplein E. coli (e.g. Qiagen QIAexpress pQE system). Recombinant polypeptidesare then normally affinity purified via Ni-NTA affinity chromatography(Qiagen). Recombinant polypeptides are then used to produce specificantibodies for example by using standard techniques for rabbitimmunization. Antibodies are affinity purified using a Ni-NTA columnsaturated with the recombinant antigen as described by Gu et al., 1994,BioTechniques 17:257-262. The antibody can than be used to screenexpression cDNA libraries to identify identical or heterologous genesvia an immunological screening (Sambrook, J. et al., 1989, “MolecularCloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press orAusubel, F. M. et al., 1994, “Current Protocols in Molecular Biology”,John Wiley & Sons).

Example 16

In Vivo Mutagenesis

In vivo mutagenesis of microorganisms can be performed by passage ofplasmid (or other vector) DNA through E. coli or other microorganisms(e.g. Bacillus spp. or yeasts such as Saccharomyces cerevisiae) whichare impaired in their capabilities to maintain the integrity of theirgenetic information. Typical mutator strains have mutations in the genesfor the DNA repair system (e.g., mutHLS, mutD, mutT, etc.; forreference, see Rupp, W. D., 1996, DNA repair mechanisms, in: Escherichiacoli and Salmonella, p. 2277-2294, ASM: Washington.) Such strains arewell known to those skilled in the art. The use of such strains isillustrated, for example, in Greener, A. and Callahan, M., 1994,Strategies 7: 32-34. Transfer of mutated DNA molecules into plants ispreferably done after selection and testing in microorganisms.Transgenic plants are generated according to various examples within theexemplification of this document.

Example 17

In Vitro Analysis of the Function of Physcomitrella Genes in TransgenicOrganisms

The determination of activities and kinetic parameters of enzymes iswell established in the art. Experiments to determine the activity ofany given altered enzyme must be tailored to the specific activity ofthe wild-type enzyme, which is well within the ability of one skilled inthe art. Overviews about enzymes in general, as well as specific detailsconcerning structure, kinetics, principles, methods, applications andexamples for the determination of many enzyme activities may be found,for example, in the following references: Dixon, M., and Webb, E. C.,1979, Enzymes. Longmans: London; Fersht, 1985, Enzyme Structure andMechanism. Freeman: New York; Walsh, 1979, Enzymatic ReactionMechanisms. Freeman: San Francisco; Price, N. C., Stevens, L., 1982,Fundamentals of Enzymology. Oxford Univ. Press: Oxford; Boyer, P. D.,ed., 1983, The Enzymes, 3^(rd) ed. Academic Press: New York; Bisswanger,H., 1994, Enzymkinetik, 2^(nd) ed. VCH: Weinheim (ISBN 3527300325);Bergmeyer, H. U., Bergmeyer, J., Graβl, M., eds., 1983-1986, Methods ofEnzymatic Analysis, 3^(rd) ed., vol. I-XII, Verlag Chemie: Weinheim; andUllmann's Encyclopedia of Industrial Chemistry, 1987, vol. A9, Enzymes.VCH: Weinheim, p. 352-363.

The activity of proteins which bind to DNA can be measured by severalwell-established methods, such as DNA band-shift assays (also called gelretardation assays). The effect of such proteins on the expression ofother molecules can be measured using reporter gene assays (such as thatdescribed in Kolmar, H. et al., 1995, EMBO J. 14: 3895-3904 andreferences cited therein). Reporter gene test systems are well known andestablished for applications in both prokaryotic and eukaryotic cells,using enzymes such as β-galactosidase, green fluorescent protein, andseveral others.

The determination of activity of membrane-transport proteins can beperformed according to techniques such as those described in Gennis, R.B., 1989, Pores, Channels and Transporters, in Biomembranes, MolecularStructure and Function, pp. 85-137, 199-234 and 270-322, Springer:Heidelberg.

Example 18

Purification of the Desired Product from Transformed Organisms

Recovery of the desired product from plant material (i.e.,Physcomitrella patens or Arabidopsis thaliana), fungi, algae, ciliates,C. glutamicum cells, or other bacterial cells transformed with thenucleic acid sequences described herein, or the supernatant of theabove-described cultures can be performed by various methods well knownin the art. If the desired product is not secreted from the cells, thecells can be harvested from the culture by low-speed centrifugation, andthe cells can be lysed by standard techniques, such as mechanical forceor sonification. Organs of plants can be separated mechanically fromother tissue or organs. Following homogenization, cellular debris isremoved by centrifugation, and the supernatant fraction containing thesoluble proteins is retained for further purification of the desiredcompound. If the product is secreted from desired cells, then the cellsare removed from the culture by low-speed centrifugation, and thesupernatant fraction is retained for further purification.

The supernatant fraction from either purification method is subjected tochromatography with a suitable resin, in which the desired molecule iseither retained on a chromatography resin while many of the impuritiesin the sample are not, or where the impurities are retained by the resinwhile the sample is not. Such chromatography steps may be repeated asnecessary, using the same or different chromatography resins. Oneskilled in the art would be well-versed in the selection of appropriatechromatography resins and in their most efficacious application for aparticular molecule to be purified. The purified product may beconcentrated by filtration or ultrafiltration, and stored at atemperature at which the stability of the product is maximized.

There is a wide array of purification methods known to the art and thepreceding method of purification is not meant to be limiting. Suchpurification techniques are described, for example, in Bailey, J. E. &Ollis, D. F., 1986, Biochemical Engineering Fundamentals, McGraw-Hill:New York. Additionally, the identity and purity of the isolatedcompounds may be assessed by techniques standard in the art. Theseinclude high-performance liquid chromatography (HPLC), spectroscopicmethods, staining methods, thin layer chromatography, NIRS, enzymaticassay, or microbiologically. Such analysis methods are reviewed in:Patek et al., 1994, Appl. Environ. Microbiol. 60:133-140; Malakhova etal., 1996, Biotekhnologiya 11:27-32; Schmidt et al., 1998, BioprocessEngineer 19:67-70; Ulmann's Encyclopedia of Industrial Chemistry, 1996,vol. A27, VCH: Weinheim, p. 89-90, p. 521-540, p. 540-547, p. 559-566,575-581, and p. 581-587; Michal, G., 1999, Biochemical Pathways: AnAtlas of Biochemistry and Molecular Biology, John Wiley and Sons;Fallon, A. et al., 1987, Applications of HPLC in Biochemistry in:Laboratory Techniques in Biochemistry and Molecular Biology, vol. 17.

APPENDIX Nucleotide sequence of the partial PK-3 from Physcomitrellapatens (SEQ ID NO:1) CGGCACCAGCATCTTCGCGAGGCATGTGATGTGTGGTCGGTGGAGTTAGCTTCTACGGGCAACTGGAAATCCAGGGAATTCTGCCAGAATTATACGTACTAAAGTAGAAATTTACGTTTCGGGGACTTCGAGTCTTCTATGGCATCTGCGACTGCGGGTATTATCAACAGCACAAACATGATCGGAGGAGGAATAGCTCCAACTAAAGCTGGCTCAAGCGGAGTAGAATTGTTACCGAAAGAAATGCACGACATGAAGCTCAGGGATGACAAGGTTGACCACAGCGACGACAAGGAAATTGAGGCTTCAATAGTAGATGGAAACGGTACCGAAACTGGCCACATCATAGCTACTACTATTGGAGGGCGAAATGGACAACCTAAGCAGACGATCAGCTATTCGGCAGAACGTGTTGTTGGCACTGGATCATTCGGGATTGTCTTCCAGGCAAAATGCATCGAAACTGGGGAGACGGTGGCTATAAAGAAAGTGTTGCAGGACAAAAGATACAAGAATCGAGAGCTGCAGATCATGCGACTGTTGGACCACCCGAATATTGTAGCTTTGAAGCATTGCTTCTTCTCGACGACGGATAAAGACGAATTGTACTTAAACCTGGTGCTGGAGTATGTACCCGAGACGGTGTATCGTATTGCAAAGCACTACAATCGCATGAATCAGCGAATGCCCCTTGTTTACGTGAAACTGTACACGTATCAGATATGCCGATCACTGGCATATATCCACAATGGCATCGGTGTCTGCCACCGCGACATCAAGCCCCAGAACCTGCTGGTGGAATCCTCATACGCACCAGCTGAAACTGTGTGATTTTGGGAAGTGCGAAAGTGCTGGTGAAAGGGGAGCCCAATATCTCGTACATTTGTTCGCGGTACTACCGTGCTCCGGGAGCTTATTTTTGGAGCGACGGAGTACACGACTGCCATAGATATATGGTCGATGGGTTGCGTGATGGCAGAGCTTCTACTAGGACAGCCTTTGTTTCCTGGAGAGAGTGGAGTGGATCAATTGGTGGAAATCATCAAGGTTTTGGGGACACCGACTCGTGAGGAGATCAAGTGCATGAATCCGAACTACAC Nucleotide sequenceof the full-length PK-3 from Physcomitrella patens (SEQ ID NO:2)GGCACCAGCATCTTCGCGAGGCATGTGATGTGTGGTCGGTGGAGTTAGCTTCTACGGGCAACTGGAAATCCAGGGAATTCTGCCAGAATTATACGTACTAAAGTAGAAATTTACGTTTCGGGGACTTCGAGTCTTCTATGGCATCTGCGACTGCGGGTATTATCAACAGCACAAACATGATCGGAGGAGGAATAGCTCCAACTAAAGCTGGCTCAAGCGGAGTAGAATTGTTACCGAAAGAAATGCACGACATGAAGCTCAGGGATGACAAGGTTGACCACAGCGACGACAAGGAAATTGAGGCTTCAATAGTAGATGGAAACGGTACCGAAACTGGCCACATCATAGCTACTACTATTGGAGGGCGAAATGGACAACCTAAGCAGACGATCAGCTATTCGGCAGAACGTGTTGTTGGCACTGGATCATTCGGGATTGTCTTCCAGGCAAAATGCATCGAAACTGGGGAGACGGTGGCTATAAAGAAAGTGTTGCAGGACAAAAGATACAAGAATCGAGAGCTGCAGATCATGCGACTGTTGGACCACCCGAATATTGTAGCTTTGAAGCATTGCTTCTTCTCGACGACGGATAAAGACGAATTGTACTTAAACCTGGTGCTGGAGTATGTACCCGAGACGGTGTATCGTATTGCAAAGCACTACAATCGCATGAATCAGCGAATGCCCCTTGTTTACGTGAAACTGTACACGTATCAGATATGCCGATCACTGGCATATATCCACAATGGCATCGGTGTCTGCCACCGCGACATCAAGCCCCAGAACCTGCTGGTGAATCCTCATACGCACCAGCTGAAACTGTGTGATTTTGGAAGTGCGAAAGTGCTGGTGAAAGGGGAGCCCAATATCTCGTACATTTGTTCGCGGTACTACCGTGCTCCGGAGCTTATTTTTGGAGCGACGGAGTACACGACTGCCATAGATATATGGTCGATGGGTTGCGTGATGGCAGAGCTTCTACTAGGACAGCCTTTGTTTCCTGGAGAGAGTGGAGTGGATCAATTGGTGGAAATCATCAAGGTTTTGGGGACACCGACTCGTGAGGAGATCAAGTGCATGAATCCGAACTACACAGAGTTCAAGTTTCCACAAATCAAGGCGCACCCGTGGCACAAAGTTTTCCACAAACGCATGCCACCTGAAGCAGTTGACTTGGTGTCAAGGCTCCTTCAGTACTCTCCAAATCTGCGGTGCAACGCTCTGGAAGCGTGTGTGCACCCGTTCTTTGATGAGCTAAGGGATCCTAACTGCCGGCTTCCGAATGGGCGGCCACTGCCCTCTCTGTTCAACTTCAAAACCCAAGAGTTGAAGGGTGCAACTCCTGATATTCTGCAGCGTTTGATACCCGAGCACGCGAGGAAGCAGAATCCGATGCTGGCGCTGTGAGGGGTGCCTGGAAAGAGATCGGAAGAGTCTACTGCGTGAAAGGTTTTCCTCTGTTTGGAGGAGTGGTCCGCTTTGTGGAGGGCTTCATAGGCACTCTGTATCATTGCTTAAACACGTAAAGTCAACCAATTTGCTATGGATCCCTGCTTTCGCTGTGATTGGAGGAAGACTTAGTAGACGATTAGCATGCCACTTTTAGGAACGGCAATTCTCCTGTAGTGAAGGTTACGATTCTATTGTACTTCAGAACGGTAAAGGTATTTAGGGGTTCTCAGTGCTTCCTGATTTGGGTACGTGATGTACCATTGGAAAGGCTTCAAACGCATGTATATCTATGAGACTTTGACGTTACTTTTTATCGTCAGTACTCAGGAAGCTCCTCTCTGGATGGGATTATCCATTCGTGCCGTTCGAATCGCAATAAAAAAAAAAAAAAAAAA Deduced amino acidsequence of PK-3 from Physcomitrella patens (SEQ ID NO:3)MASATAGIINSTNMIGGGIAPTKAGSSGVELLPKEMHDMKLRDDKVDHSDDKEIEASIVDGNGTETGHIIATTIGGRNGQPKQTISYSAERVVGTGSFGIVFQAKCIETGETVAIKKVLQDKRYKNRELQIMRLLDHPNIVALKHCFFSTTDKDELYLNLVLEYVPETVYRIAKHYNRMNQRMPLVYVKLYTYQICRSLAYIHNGIGVCHRDIKPQNLLVNPHTHQLKLCDFGSAKVLVKGEPNISYICSRYYRAPELIFGATEYTTAIDIWSMGCVMAELLLGQPLFPGESGVDQLVEIIKVLGTPTREEIKCMNPNYTEFKFPQIKAHPWHKVFHKRMPPEAVDLVSRLLQYSPNLRCNALEACVHPFFDELRDPNCRLPNGRPLPSLFNFKTQELKGATPDILQRLIPEHARKQNPMLAL* Nucleotide sequence of the partial PK-4 fromPhyscomitrella patens (SEQ ID NO:4)GCACGAGGATCGACCGGGTGGAGTACGTGCACTCGCGAGGTCTAATTCATCGTGACTTGAAACCAGATAATTTCCTCATGGGCTGCGGCCGGCAAGGGAACCAAGTGTTCATTATTGACTTTGGCTTGGCAAAAGAGTACATCGACCCCGCGACACGTAGACACATTCCTTACCGAGATAGAAAGAGCTTTACAGGAACAGCGCGGTATGCTAGTAGGAATCNCCACNAAGGAATCGAACACAGCAGGAGAGATGACATANAATCNCTTGGTTACATTCTTATGTACTTTCTTAGGGGGAATTTACCATGGCAAGGTCAAGGGGGGCAACGTTTCACCGATCAGAAGCAACATGAGTACATGCNCAACAAAATTAAGATGGAGACTANCATCNAGGATCT CTGCGATGGGTACCCAGACANucleotide sequence of the full-length PK-4 from Physcomitrella patens(SEQ ID NO:5) GCCCTTATCCCGGGAGGCATTGAACTACCTGGAGTGAGATTTTTTTGGGAATTTGAAAGAGAATTACATATATACAAGGTTGAGGCTCACCGAGAACAAGTCTGCTGATAGCTTCTTCACTCTTGAAATAGATAGTTCATCATGGATTCAGGAGGTGACCGCGTGCGAGCTCCTCAGAAGCAGTCTCGCGAGGAGGATCAGTACCGTTCATTGAACATTGCTACAGAGCATCGTCAGCATATACAGAAGCACCAACAACACCAACAGCAGCCGGGGACTGGATTGGTTGTTGAAACGCTTCAAAAAACACTATGTAACGTGACTGTGACCTCACCTACAAGCAGTCCGGAGGGGGGTAGATTACGTACTGTTGCGAACAAGTATGCAGTGGAAGGAATGGTCGGCAGTGGCGCATTTTGCAAGGTGTACCAGGGTTCTGACTTAACCAACCATGAGGTTGTGGGCATCAAGCTCGAGGATACAAGAACAGAGCACGCACAATTGATGCACGAGTCGCGATTATACAACATTTTGCGGGGTGGAAAGGGAGTGCCCAACATGAGATGGTTTGGGAAAGAGCAAGACTACAATGTGATGGTGCTAGATTTGCTGGGGCCTAACCTACTGCACCTTTTCAAGGTGTGTGGGCAAAGATTTTCGTTGAAGACGGTGATCATGTTGGGGTACCAAATGATCGACCGGGTGGAGTACGTGCACTCGCGAGGTCTAGTTCATCGTGACTTGAAACCAGATAATTTCCTCATGGGCTGCGGCCGGCAAGGGAACCAAGTGTTCATTATTGACTTTGGCTTGGCAAAAGAGTACATCGACCCCGCGACACGTAGACACATTCCTTACCGAGATAGAAAGAGCTTTACAGGAACAGCGCGGTATGCTAGTAGGAATCAGCACAAAGGAATCGAACACAGCAGGAGAGATGACATAGAATCACTTGGTTACATTCTTATGTACTTTCTTAGGGGGAATTTACCATGGCAAGGTCAAGGGGGGCAACGTTTCACCGATCAGAAGCAACATGAGTACATGCACAACAAAATTAAGATGGAGACTACCATCGAGGATCTCTGCGATGGGTACCCCAGACAATTTGCCGACTTTTTACACCACGCGCGCGAGTTGGGATTCTATGAGCAGCCTGACTACTCGTACCTTCGCAGCCTGTTCCGTGATCTTTTCATTCAGAAGAAATTCCAGCTTGACCATGTCTACGACTGGACAGTGTACACTCAACCTCCTCAGAATGGCTCTGCACAAACAGTTCGAAGCCCGGCTGCCGGTCCACAGACTCACTTACAAAGTCGCCCTTCCAATGTATCATATTGTCCACCTCTGACTAAACCAGAGTTCCGGCGTGAGGTAGTTGCGGCGAATTAGGGTTTACACAGGAAGAGATGTGGTAAAGCATCTCATCTTCTTCGTTCTGGTGCCAAAATGGTACAAGGTCGTCTGCTGTCTCTTTCTCGCAAGCCCTCACATATAGATGAAGGTTTGTGAAGTTAGAGATGCAACTACCAAGCAAAGGCTAGGAAAAGAGCTGTAGACTTTCTAGTGTGTAGTGTGTAAATCAAGGCTTCTGGCATGGTATCGGCAGTCAGGTGCATGGAGCAGAATAGAAATTACTTCGTGCATGACAAGATTTTTTTTCTTGCAGAGCTCTCGACGGTTCTGCGATCTCACTTCTCTACACAACCAGCGCTCCTTTAATTGAAAAGAGGATCTGGTACGAGTATGATAAAGTTAACACAGATTACTAGTTCAACGATATCGCAAGGGC Deduced amino acidsequence of PK-4 from Physcomitrella patens (SEQ ID NO:6)MDSGGDRVRAPQKQSREEDQYRSLNIATEHRQHIQKHQQHQQQPGTGLVVETLQKTLCNVTVTSPTSSPEGGRLRTVANKYAVEGMVGSGAFCKVYQGSDLTNHEVVGIKLEDTRTEHAQLMHESRLYNILRGGKGVPNMRWFGKEQDYNVMVLDLLGPNLLHLFKVCGQRFSLKTVIMLGYQMIDRVEYVHSRGLVHRDLKPDNFLMGCGRQGNQVFIIDFGLAKEYIDPATRRHIPYRDRKSFTGTARYASRNQHKGIEHSRRDDIESLGYILMYFLRGNLPWQGQGGQRFTDQKQHEYMHNKIKMETTIEDLCDGYPRQFADFLHHARELGFYEQPDYSYLRSLFRDLFIQKKFQLDHVYDWTVYTQPPQNGSAQTVRSPAAGPQTHLQSRPSNVSY CPPLTKPEFRREVVAAN*Nucleotide sequence of the partial PK-10 from Physcomitrella patens (SEQID NO:7) GCACGAGCGCACTTGGTTTCTGCCACTTATTCCAGCTGGTAAAGAAAAACCACCTAAAATGAAAGTGTTTGAAGCAGATACATTTGAGAAGGAAGTGGAAGAACCGAAGATCAAGGCCTTACCTCCATTGAAGTCACTTAAAGTACCTCCAGCTTTGAAGGTTGAGGAAGCTACCTACAAGGTTGAAAGTGAAGGGAAGGTGAACAAGAGCAACATTACAGCAAGAGAGTTTTCCGTCGCAGAACTTCAGGCGGCTACGGACAGTTTCTCAGAGGATAATTTACTTGGCGAAGGTTCGCTTGGTTGTGTTTACCGCGCGGAGTTCCCCGACGGTGAGGTTCTAGCTGTCAGAAACTTGATACAACAGCCTCCATGGTTCGGAATGAAGATGATTTCTTGAGCGTTGTCGATGGCTTGGCCCGGCTACAATACCAATTCTAATGAACTCGTAGGCTACTGTGCCGAGCATGGGCAACGACTTCTGGTCTACAAGTTCATCAGTCGAGGGACACTCCATGAACTGCTTCATGGCTCAGCCG Nucleotide sequence of thefull-length PK-10 from Physcomitrella patens (SEQ ID NO:8)TTTCTGGAATAGCTCAGAAGCGTTGCAAAATTTATCAGGAGGTTTGCAGACATGGTGATGAGGAAAGTGGGCAAGTATGAAGTGGGGCGAACTATTGGTGAGGGAACCTTCGCCAAGGTGAAATTTGCCCAGAACACCGAGACAGGGGAGAGCGTGGCCATGAAGGTGCTAGATCGTCAGACGGTGCTCAAGCACAAGATGGTAGAGCAGATCAGGCGAGAAATATCCATAATGAAGCTGGTTAGGCATCCTAATGTTGTCCGATTGCACGAGGTTCTGGCAAGTCGTTGCAAGATTTACATCATTTTGGAGTTTGTAACGGGCGGGGAGCTTTTTGACAAAATTGTGCATCAAGGAAGGCTTAATGAGAACGACTCTCGCAAATATTTTCAGCAGCTCATGGATGGAGTTGATTATTGCCACAGCAAGGGCGTCTCACATCGAGATTTGAAGCCTGAAAATCTCCTTCTTGATTCACTGGACAATCTCAAAATATCAGATTTTGGTCTGAGTGCTCTTCCTCAGCAAGTGAGGGAAGATGGACTTTTGCACACCACTTGTGGTACTCCCAATTATGTTGCACCTGAGGTTCTTAATGATAAGGGCTACGATGGTGCAGTGGCTGATATCTGGTCTTGCGGTGTCATCTTGTTTGTATTAATGGCTGGATTTCTCCCATTTGATGAGGCTGACTTGAATACTCTTTACAGCAAGATACGAGAGGCAGATTTTACTTGTCCACCTTGGTTTTCCTCCGGCGCCAAAACACTGATTACTAATATTCTGGATCCCAATCCCCTAACACGTATCAGGATGAGAGGAATTCGGGATGACGAATGGTTCAAAAAGAACTATGTTCCTGTTCGTATGTATGACGATGAAGATATTAATCTTGATGATGTGGAGACTGCTTTTGATGATTCTAAGGAACAATTTGTGAAAGAGCAGAGGGAGGTGAAAGACGTGGGTCCGTCGTTGATGAATGCCTTTGAACTCATAAGCCTATCTCAAGGACTAAACCTCTCTGCGTTGTTTGATAGACGTCAGGACCATGTAAAGCGCCAAACTCGTTTCACTTCAAAGAAACCAGCTCGAGATATAATTAATAGAATGGAAACCGCTGCGAAGTCGATGGGCTTTGGTGTTGGAACGCGTAACTACAAGATGAGACTCGAGGCAGCTAGTGAGTGCAGAATATCACAGCACTTGGCTGTGGCTATCGAAGTGTACGAGGTGGCTCCTTCTTTATTCATGATTGAAGTGCGGAAGGCTGCGGGTGATACTTTGGAATATCACAAGTTCTATAAAAGCTTTTGTACCCGGTTGAAAGATATCATATGGACAACGGCAGTTGATAAGGACGAAGTTAAGACATTGACGCCATCTGTAGTTAAGAATAAATAATTCTGCTCCAGCATTAACTTGGATGAGGAGCAAGGATATACCGCTG CATCGAGCTCCGAAGGGCDeduced amino acid sequence of PK-10 from Physcomitrella patens (SEQ IDNO:9) MVMRKVGKYEVGRTIGEGTFAKVKFAQNTETGESVAMKVLDRQTVLKHKMVEQIRREISIMKLVRHPNVVRLHEVLASRCKIYIILEFVTGGELFDKIVHQGRLNENDSRKYFQQLMDGVDYCHSKGVSHRDLKPENLLLDSLDNLKISDFGLSALPQQVREDGLLHTTCGTPNYVAPEVLNDKGYDGAVADIWSCGVILFVLMAGFLPFDEADLNTLYSKIREADFTCPPWFSSGAKTLITNILDPNPLTRIRMRGIRDDEWFKKNYVPVRMYDDEDINLDDVETAFDDSKEQFVKEQREVKDVGPSLMNAFELISLSQGLNLSALFDRRQDHVKRQTRFTSKKPARDIINRMETAAKSMGFGVGTRNYKMRLEAASECRISQHLAVAIEVYEVAPSLFMIEVRKAAGDTLEYHKFYKSFCTRLKDIIWTTAVDKDEVKTLTPSVVKN K* Nucleotide sequenceof the partial PK-11 from Physcomitrella patens (SEQ ID NO:10)GGCACGAGATTTGGTTGCAAAATAGGTAACTACAACTTAAGAAGAAAAACAATCTCTCTCTTTCCCCACACAAGATACAACTTCGCTTTTTCCATCACTTACACCAGAAAGCCCAAAGTAGGGTAGATTGTCACACATCGCTATGATCCCAATTAAGCATCTACTACTTTTCATCAGATCAGCAAACTACCAATCATAGAAACTAGGTGATGAATATTACGATACTTTCAGGTTCAATGCGAAATCCAAGGTTAACAGTAATGAATGTATTCAAGCTCTGTACATGCATTAATTTTATGCTACCAGTAGAAAACTTCATTTGACGATGCAGCGGTATATCCTTGCTCCTCATCCAAGTTAATGCTGGAGCAGAATTATTTATTCTTAACTACAGATGGCGTCAATGTCTTAACTTCGTCCTTATCAACTGCCGTTGTCCATATGATATCTTTCAACCGGGTACAAAAGCTTTTATAGAACTTGTGATATTCCAAAGTATCACCCGCAGCCTTCCGCACTTCAATCATGAATAAAGAAGGAGCCACCTCGTACACTTCGATAGCCCAGCCAAGTGCTGTGATATTCTGCCTCACTACTGCC TCGAGC Nucleotidesequence of the full-length PK-11 from Physcomitrella patens (SEQ IDNO:11) ATCCCGGGTGTCGGAATTCGGTCACAATGAGCTAGTGTGTTGTTTGATTGTGGCCTCAGCTGGAGAGGCTTTGGTATCGTTAGCAGCGAGTGACGCTGTTGAAGGATTGTATCCATCCACAAGCGAGAAGCCTTGCCTAATTTTTGGGAGGGAAAGGTGGTTCTCACATGAGAGGAGCAGTTGTCGATGCCCCAATGAAGGGTGACAGGAGAGCATGCATTTTGGGAGGAATGGGAAGACCTAATGGTGGAACCATCTTGTACGTGTTGGTGATTTCATTCATTGCTTTGGTGAATGGAGCCACCGATCCGAACGATGTGTCTGCTTTGAATACTATGTTCACTGGCTTCAACAGCGATCCTAAGCTCACGAACTGGGTGCAAAACGCGGGTGATCCCTGCGGAACCAACTGGCTGGGCGTTACTTGTGATGGGACCTTCGTCACCTCAATCAAGCTATCCAACATGGGACTGAATGGGAAGGTGGAGGGATGGGTGTTGCAGAAGTTTCAACACCTCTCTGTGCTTGACCTTAGCCATAATAATCTTGCTAGCGGAATTCCTGAGATGTTTCCTCCCAAGTTGACTGAACTAGATTTGTCTTACAACCAGCTCACGGGTAGTTTTCCTTATTTGATAATCAACATCCCTACTTTGACAAGCATAAAACTGAATAACAACAAGCTGAGTGGAACGCTCGATGGGCAGGTTTTCAGTAAACTCACAAACTTAATCACCCTCGATATTTCCAACAACGCAATTACAGGGCCGATTCCCGAGGGCATGGGTGACATGGTCAGCCTAAGATTTTTGAACATGCAAAATAATAAGCTGACTGGACCAATCCCAGACACATTGGCTAATATTCCATCTCTAGAAACATTGGACGTATCTAACAACGCGCTTACTGGCTTTCTCCCACCAAACCTGAACCCAAAGAATTTCAGATATGGAGGCAATCCACTCAACACCCAAGCCCCTCCTCCACCACCGTTTACACCACCGCCACCTTCAAAGAATCCAAAGCCTATTCCTCCTCCACCCCACCCTGGTAGCCGAACACCAGATACTGCTCCTAAGGCTGAAGGCGGCATCGTATCAGGCGCAGCAATTGCTGGGATTGTCGTGGGAGCAATTTTGGTGCTTGCAGCAATTTTCATAGCTGTATGGTTCTTTGTCGTCCGTAAAAGATCTGAGCTTACCAAACCTTTGGATTTAGAGGCTAATCACAGCAGCCGACGCACTTGGTTTCTGCCACTTATTCCAGCTGGTAAAGAAAAACCACCTAAAATGAAAGTGTTTGAAGCAGATACATTTGAGAAGGAAGTGGAAGAGCCGAAGATCAAGGCCTTACCTCCATTGAAGTCACTTAAAGTACCTCCAGCATTGAAGGTTGAGGAAGCTACCTACAAGGTTGAAAGTGAAGGGAAGGTGAACAAGAGCAACATTACAGCAAGAGAGTTTTCCGTCGCAGAACTTCAGGCGGCTACGGACAGTTTCTCAGAGGATAATTTACTTGGCGAAGGTTCGCTTGGTTGTGTTTACCGCGCGGAGTTCCCCGACGGTGAGGTTCTAGCTGTCAAGAAACTTGATACAACAGCCTCCATGGTTCGGAATGAAGATGATTTCTTGAGCGTTGTCGATGGCTTGGCCCGGCTACAACATACCAATTCTAATGAACTCGTAGGCTACTGTGCCGAGCATGGGCAACGACTTCTGGTCTACAAGTTCATCAGTCGAGGGACACTCCATGAACTGCTTCATGGCTCAGCCGATAGCCCCAAGGAGTTGTCATGGAATGTCCGTGTGAAGATTGCACTTGGTTGTGCGCGGGCTCTTGAGTATTTCCATGAAATCGTTTCGCAGCCGGTTGTGCACCGCAACTTTAGATCCTCAAACATTCTTTTGGATGATGAGCTGAACCCACATGTGTCGGATTGTGGTTTGGCTGCTTTTACCCCATCCAGTGCTGAACGGCAGGTCTCTGCCCAAGTGTTGGGATCTTTTGGACACAGTCCCCCTGAATTCAGCACATCTGGAATGTATGATGTGAAAAGCGACGTTTATAGCTTTGGTGTTGTGATGCTTGAGCTTATGACAGGACGCAAGCCTTTAGACAGCTCAAGACCAAGATCCGAGCAAAACCTGGTGCGATGGGCAACACCACAACTGCATGATATTGATGCACTCGCAAGAATGGTGGATCCAGCGTTAGAGGGTGCTTACCCTGCCAAGTCCCTCTCCCGGTTCGCCGACATCGTTGCCTTGTGTGTCCAGCCCGAACCCGAATTCCGACCTCCTATATCTGAAGTAGTGCAGTCCCTGGTAAGGCTTATGCAGCGTGCAGCTTTAAGTAAACGCCGGCATGAGTACAACGCAGGCGTTCCTCAGACTGATATGGAGGACCCTAGTGATTACTTGTGACAGAAGTAAGTATCCTGGTCGATACTTCCCAATTTCAAGCATAGAGAACCTCCCGCGCGTCTACTCCCACTTGATTTTCAAAGCTGGCGAAAAGTGGCCAAATTTGTGGATTTGTGACACCTTGCAACTAAATCGGGGAGATATTCAGCTTCTTTGCAATTCCAGACCATGATGGCACAGACTTTGGCTTGCATCCTCCTCATTATTACTGAAGCTTTTGCTTCTAATGGCGGATTACTGATTATGGATGACTATCCCGTTTCCAGGCAGACGTGAAGAGAAGTGTTGGCTTCCGAAGTTGTTAAATTGTATCGACGGCTGAAAGCTTTTTTAAGAGCTTACTTCTGGGTCCTAGTTAGTGATATTAAGGTCCCTGTGCCTTAAGAGTAATGTGCAATTCCTGTTGTGTTGCAAACTCGGGTAACGCTTTGTCTTGTAGTTTTGGCACATTACAAGGTTAGTTCGACAGTGAACTCACAATTTGAACAGATTAGTTAGGGAGTGTAACTCTAGCAAAAGTTGATTCCTTGTGGTTACCCAATTTTTTGAATGTGAACTCCCACTCATTGGTGTGATGGAGTACATGATTCGCACGAGCTCGC Deduced amino acid sequence of PK-11 fromPhyscomitrella patens (SEQ ID NO:12)MRGAVVDAPMKGDRRACILGGMGRPNGGTILYVLVISFIALVNGATDPNDVSALNTMFTGFNSDPKLTNWVQNAGDPCGTNWLGVTCDGTFVTSIKLSNMGLNGKVEGWVLQKFQHLSVLDLSHNNLASGIPEMFPPKLTELDLSYNQLTGSFPYLIINIPTLTSIKLNNNKLSGTLDGQVFSKLTNLITLDISNNAITGPIPEGMGDMVSLRFLNMQNNKLTGPIPDTLANIPSLETLDVSNNALTGFLPPNLNPKNFRYGGNPLNTQAPPPPPFTPPPPSKNPKPIPPPPHPGSRTPDTAPKAEGGIVSGAAIAGIVVGAILVLAAIFIAVWFFVVRKRSELTKPLDLEANHSSRRTWFLPLIPAGKEKPPKMKVFEADTFEKEVEEPKIKALPPLKSLKVPPALKVEEATYKVESEGKVNKSNITAREFSVAELQAATDSFSEDNLLGEGSLGCVYRAEFPDGEVLAVKKLDTTASMVRNEDDFLSVVDGLARLQHTNSNELVGYCAEHGQRLLVYKFISRGTLHELLHGSADSPKELSWNVRVKIALGCARALEYFHEIVSQPVVHRNFRSSNILLDDELNPHVSDCGLAAFTPSSAERQVSAQVLGSFGHSPPEFSTSGMYDVKSDVYSFGVVMLELMTGRKPLDSSRPRSEQNLVRWATPQLHDIDALARMVDPALEGAYPAKSLSRFADIVALCVQPEPEFRPPISEVVQSLVRLMQRAALSKRRHEYNAGVPQTDMEDPSDY L* Nucleotidesequence of BnPK-1 from Brassica napus (SEQ ID NO:13)AACAAAAAAAAATCTAAGGTTTATCTTTTTCTTCTTCTATCTGATCATCAATCATCGAGAGAGAAAAAAGTATACTTTTTTAGATGTGAAGAAGCTCATCAATCGAAGAAGACAATCATCAAATGCTTCACTTTGGTTCCCTTTCTTCATCAGAAAACTCGAGGTAGATCAGTTCTTTGATGGGATGGGACACCAAATCGCTAAGTGTTATGATACCAGCAACTACTAGTTACGTGCTATCTCCAGAGCAAATACCATGGCTTCAAACGGAGTAGGCAGTTCGAGATCTTCCAAAGGTGTGAAGGCCTCTTCTAGCTCAGTCGATTGGTTGACCAGAGATTTGGTTGAGATGAGGATAAGGGACAAGGTCGAGACTGATGATGAGAGGGATAGTGAACCAGATATTATTGATGGCGCTGGCACTGAACCTGGCCATGTGATTAGAACCACAGTCCGTGGACGCAATGGTCAATCAAGACAGACAGTCAGTTACATATCAGAGCATGTAGTTGGTACTGGTTCCTTTGGCATGGTTTTTCAAGCCAAGTGTAGGGAAACTGGGGAGATTGTTGCAATCAAGAAGGTTCTACAAGACAAGCGTTACAAGAACAGGGAGCTACAAATTATGCAGATGCTAGACCACCCCAATGTCGTTGCTCTAAAGCATAGCTTCTACACGAGAGCTGATAACGAAGAGGTTTATTTGAATCTTGTCCTTGAGTTTGTGCCTGAGACCGTCAATAGGGCTGCAAGAAGTTACACTAGGACGAACCAGCTAATGCCTTTAATATACGTTAAACTCTACACCTATCAGATTTGCAGGGCGCTTGCTTACATCCATAATTGCTTTGGTCTTTGTCACCGTGATATTAAGCCTCAAAACTTGCTAGTGAACCCACATACGCATCAGCTGAAAATCTGTGACTTCGGGAGTGCAAAAGTGTTGGTGAAAGGAGAACCCAATGTTTCTTACATCTGTTCTAGATACTATCGTGCTCCAGAACTCATTTTTGGCGCCAGCGAATACACACCTGCAATTGATATATGGTCAACTGGTTGTGTGATGGCTGAATTGCTTCTTGGACAGCCTCTGTTCCCTGGTGAAAGCGGAGTCGATCAGCTTGTTGAAATCATTAAGGTTTTAGGTACACCAACGAGGGAGGAAATCAAGTGCATGAATCCAAACTATACAGAATTTAAATTCCCCCAGATAAAACCTCACCCATGGCACAAGGTCTTCCAAAAACGTTTACCGCCAGAAGCGGTTGATCTTCTATGTAGGTTCTTCCAATATTCCCCTAATCTGAGATGCACAGCTTTGGAAGCGTGTATTCATCCGTTATTTGATGAGCTAAGGGACCCGAACACTCGTCTTCCCAATGGCCGGCCACTTCCTCCGCTTTTCAACTTCAAACCTCAAGAGCTATCTGGCATCCCTTCTGAAATCGTGAACAGGCTTGTACCAGAACATGCCCGTAAGCAGAACTTCTTCATGGCGTTGGATGCCTAAGCGCTTATCCTGTTTCTTTTCTTTTTCTTGCTTATGTATAAACTCTCTAGATATCGGGTATTTGGAGCAGCCAGAAGGCATTACACGCCCTCTTTGGCTTTTTTTTATCAGTGAGTTGTTTGGTTATCGGGACACGATGATGCATGAATACAAACAGTACTTGAGGTCGCTGCTGGCTTATAAGACCACTTGTTTGTTTCACAACCAGTTCTTATATATATTATTATACAAAAAAAAAAA AAAAAAA Deduced aminoacid sequence of BnPK-1 from Brassica napus (SEQ ID NO:14)MASNGVGSSRSSKGVKASSSSVDWLTRDLVEMRIRDKVETDDERDSEPDIIDGAGTEPGHVIRTTVRGRNGQSRQTVSYISEHVVGTGSFGMVFQAKCRETGEIVAIKKVLQDKRYKNRELQIMQMLDHPNVVALKHSFYTRADNEEVYLNLVLEFVPETVNRAARSYTRTNQLMPLIYVKLYTYQICRALAYIHNCFGLCHRDIKPQNLLVNPHTHQLKICDFGSAKVLVKGEPNVSYICSRYYRAPELIFGASEYTPAIDIWSTGCVMAELLLGQPLFPGESGVDQLVEIIKVLGTPTREEIKCMNPNYTEFKFPQIKPHPWHKVFQKRLPPEAVDLLCRFFQYSPNLRCTALEACIHPLFDELRDPNTRLPNGRPLPPLFNFKPQELSGIPSEIVNR LVPEHARKQNFFMALDA*Nucleotide sequence of BnPK-2 from Brassica napus (SEQ ID NO:15)TTTTCTCTCTCTCTCTCTCTCTCCACATTTGATGATCATTACCAACCAAACTAATTGAAATCCATTTGTTCTCTCTCTCTCTCTCTCTCTCTCACACTCTCTTCTCTGCTCTTCTCTGCGCCTCTAACGTCATGGCTGACGATAGGGAGATGCCGCCGGCTGCTGTAGTTGATGGACATGACCAAGTCACTGGCCACATAATCTCCACCACCATCGGTGGTAAAAACGGAGAACCAAAACAGACAATAAGTTACATGGCGGAGCGAGTTGTCGGTACAGGCTCCTTCGGGATAGTGTTCCAGGCGAAGTGTCTGGAGACTGGAGAAACCGTGGCGATAAAGAAGGTTTTGCAAGACAGGAGGTACAAGAACCGAGAGCTTCAGCTGATGCGTGTGATGGACCATCCGAATGTTGTTTGTTTGAAGCATTGCTTCTTCTCGACCACGAGCAAAGACGAGCTGTTTCTGAACTTGGTTATGGAGTATGTCCCTGAGAGCTTGTACCGAGTTCTGAAACATTACAGCACTGCTAACCAGAGGATGCCGCTTGTTTATGTTAAACTCTATATGTACCAGATCTTCAGAGGACTTGCTTACATTCACAATGTTGCTGGAGTTTGTCACAGAGATCTAAAGCCTCAAAATCTTCTGGTTGATCCTCTGACTCATCAAGTGAAGATCTGTGATTTTGGCAGTGCGAAACAGCTTGTTAAAGGTGAAGCCAACATCTCTTACATATGTTCAAGATTCTACCGTGCACCTGAACTTATATTCGGTGCCACTGAGTACACAACTTCCATTGATATTTGGTCTGCTGGTTGTGTTCTCGCTGAGCTTCTTCTTGGTCAGCCACTATTCCCTGGAGAAAATGCTGTGGGTCAGCTCGTTGAAATCATCAAAGTTCTTGGTACACCAACTCGAGAAGAGATCCGTTGTATGAATCCACACTACACAGACTTTAGGTTCCCGCAGATAAAGGCACATCCTTGGCACAAGATTTTCCACAAAAGGATGCCTCCAGAAGCCATTGATTTTGCATCAAGGCTGCTTCAGTACTCTCCAAGTCTTAGATGCACAGCGCTTGAAGCTTGTGCACATCCGTTCTTTGATGAGCTTAGAGAACCAAATGCTCGTTTACCAAACGGACGGCCTTTCCCGCCGCTCTTCAACTTCAAACAAGAGGTAGCTGGAGCTTCACCTGAGCTGGTCAACAAGTTGATTCCAGACCATATCAAGACGCAGTTGGGTCTAAGCTTCTTGAATCAGTCTGGAACTTAAACAAACGATCAAAAAGACAAGAACTTTTTTATATATAATTGTACCATTACTCAGAACCAGAAGAAGGTTAGTTGAAGGCACGTGGAGGACACAGTTAGAGGTTTTGCCTCCTCAAAACTCGTTCCAGGAATGAAGGTCAAAAAAGACAAGCTTCTCTACAACCTGACTTCCCCCAAGCCTGCAAGAAAAGCTACTCAGTTGTATCTTCTTCTTCTTCTTTTGTCCTTTTTTAAAAATGTTTGGTTAAAGCAAAGAACAAAATCTTCTCTTTTTGCTTTATTCTTACTGCATCTGTAAATGAGTTTAGTCAGAGATTTTTATAT AGTAAAAAAAAAAAAAAAAAADeduced amino acid sequence of BnPK-2 from Brassica napus (SEQ ID NO:16)MADDREMPPAAVVDGHDQVTGHIISTTIGGKNGEPKQTISYMAERVVGTGSFGIVFQAKCLETGETVAIKKVLQDRRYKNRELQLMRVMDHPNVVCLKHCFFSTTSKDELFLNLVMEYVPESLYRVLKHYSTANQRMPLVYVKLYMYQIFRGLAYIHNVAGVCHRDLKPQNLLVDPLTHQVKICDFGSAKQLVKGEANISYICSRFYRAPELIFGATEYTTSIDIWSAGCVLAELLLGQPLFPGENAVGQLVEIIKVLGTPTREEIRCMNPHYTDFRFPQIKAHPWHKIFHKRMPPEAIDFASRLLQYSPSLRCTALEACAHPFFDELREPNARLPNGRPFPPLFNFKQEVAGASPELVNKLIPDHIKTQLGLSFLNQSGT* Nucleotide sequence of BnPK-3 fromBrassica napus (SEQ ID NO:17)CGTCGTCGTCTCTCTCTCTTTCTTTCTCTTCTCCGTGAATCATCATCATCATCATCATCTTCGTGTTTTCTCGTTAAGCCCATTTTGTTTTTTTTTTTTCTCTGGGGAAAAACTCGGCTCAAAACGATGAATGTGATGCGTAGATTGACGAGTATCGCTTCTGGACGCGGTTTCGTCTCTTCTGATAACGTAGGAGAGACCGAGACGCCGAGATCGAAGCCTAACCAAATTTGTGAAGAGATAGAAGAGACTACACGAGAAGACTCTGTTTCTAAAACAGAGGATTCTGATTCATTACCAAAAGAGATGGGAATCGGTGATGACGACAAGGATAAGGACGGTGGGATTATCAAGGGTAATGGGACAGAGTCTGGTCGGATCATTACCACCACAAAGAAGGGTCTGAACGATCAAAGAGACAAGACAATCTCGTACAGAGCTGAACATGTGATTGGCACTGGCTCATTCGGTGTTGTCTTTCAGGCTAAGTGCTTAGAGACAGAAGAAAAAGTAGCTATCAAGAAAGTGTTGCAAGACAAGAGATACAAGAACAGAGAGCTTCAGATCATGCGGATGCTTGATCATCCTAATGTTGTTGACCTCAAGCATTCTTTCTTCTCCACCACTGAGAAAGATGAGCTTTATCTTAACCTTGTTCTTGAGTATGTACCTGAGACTATATACCGTTCTTCAAGATCTTACACCAAGATGAATCAACACATGCCCTTGATCTATATTCAGCTCTATACATATCAGATTTGCCGCGCAATGAACTATCTACATAGAGTTGTTGGAGTGTGTCACCGTGACATTAAACCTCAGAATCTATTGGTCAATAATGTTACACATGAGGTGAAGGTATGCGATTTTGGGAGCGCCAAGATGCTGATTCCGGGAGAACCCAATATATCTTACATATGCTCAAGGTATTACAGAGCTCCTGAACTCATATTTGGGGTAACTGAGTACACAACCGCCATCGATATGTGGTCTGTTGGCTGTGTCATGGCTGAACTTTTTCTTGGACATCCTCTGTTCCCTGGAGAGACTAGTGTTGATCAATTGGTTGAGATCATTAAGATTTTGGGAACACCAGCAAGAGAAGAGATCAGAAACATGAATCCTCGTTACAATGATTTTAAGTTCCCTCAGATCAAAGCTCAGCCATGGCACAAGATTTTCCGGAGACAGGTATCTCCAGAAGCAATGGATCTTGCCTCTAGACTCCTCCAGTACTCACCAAACCTGAGATGTTCAGCGCTTGAAGCATGTGCACACCCCTTCTTCGATGATCTGAGAGACCCGAGAGCATCCTTGCCTAATGGAAGAGCACTTCCTCCACTGTTTGATTTCACAGCTCAAGAACTGGCTGGTGCATCTGTTGAATTGCGTCATCGCTTAATCCCTGAACATGCAAGGAAATAACTTACTTTGTCTAACGAGACCGCTTCTTCTCTACACAGATGTTGATATCTAAATTCCTTTTTTTTTGGCATTGTTCTGGTTATGAACACCCTCATTGACCTCTGCAACCACCTTGCACTAGCAGTTCCAAAAGTGTATGATTTGTTAAGTTTGTAACTTTGTAGACTCCATTGTTGCAGACAGAAAATGCAGAATTTTCCGAGTTTGTCTCAAAAAAAAAAAAAA AAAA Deduced aminoacid sequence of BnPK-3 from Brassica napus (SEQ ID NO:18)MNVMRRLTSIASGRGFVSSDNVGETETPRSKPNQICEEIEETTREDSVSKTEDSDSLPKEMGIGDDDKDKDGGIIKGNGTESGRIITTTKKGLNDQRDKTISYRAEHVIGTGSFGVVFQAKCLETEEKVAIKKVLQDKRYKNRELQIMRMLDHPNVVDLKHSFFSTTEKDELYLNLVLEYVPETIYRSSRSYTKMNQHMPLIYIQLYTYQICRAMNYLHRVVGVCHRDIKPQNLLVNNVTHEVKVCDFGSAKMLIPGEPNISYICSRYYRAPELIFGVTEYTTAIDMWSVGCVMAELFLGHPLFPGETSVDQLVEIIKILGTPAREEIRNMNPRYNDFKFPQIKAQPWHKIFRRQVSPEAMDLASRLLQYSPNLRCSALEACAHPFFDDLRDPRASLPNGRALPPLFDFTAQELAGASVELRHRLIPEHARK* Nucleotide sequence of BnPK-4 fromBrassica napus (SEQ ID NO:19)GTTTTGGCATCTGGAGAGGGAGAGAGAGAGAGAGAAAGGGGAATAAGATGATGGAGAATCGAGTGGTGGTGGTGGCTGCTCTGTTTGCGGTCTGCATTGTAGGATTTGAGTTTAGCTTCATCCATGGAGCCACTGATGCATCAGACACTTCAGCATTGAACATGTTGTTCACCAGTATGCATTCACCAGGACAGTTAACACAATGGACTGCATCAGGTGGGGATCCTTGTGTTCAGAACTGGAGAGGCGTTACTTGCTCCAAATCACGAATTACTCAATTAAAGTTATCAGGTCTTGAGCTCTCTGGAACACTTGGGTACATGCTTGATAAATTGACTTCTCTTACAGAGCTTGATCTAAGCAGCAATAATCTTGGAGGTGATTTACCATATCAGCTTCCTCCAAATCTGCAACGGTTGAATCTTGCAAACAACCAATTCACTGGAGCTGCTCAATACTCCATTTCTAATATGGCATCACTTAAGTATCTTAATCTTGGTCACAACCAGTTTAAGGGGCAAGTAGCTGTGGACTTCTCCAAGCTCACCTCTCTTACAACCTTGGACTTCTCTTTCAACTCTTTCACATCGTCTCTACCGGGAACTTTTACTTCTCTTACAAGTTTAAAGTCCCTATACCTTCAGAACAATCAGTTCTCAGGAACACTCAATGTATTAGCCGGTCTTCCTCTTGAGACCCTGAACATTGCAAACAATGACTTCACCGGCTGGATCCCCAGTACCTTAAAGGGTACTAATTTAATAAAAGATGGTAACTCGTTCAATAATGGACCTGCACCACCACCACCACCTGGTACACCTCCAATCCACCGCTCACCGAGCCATAAATCCGGAGGAGGTTCAAACCGTGATTCTACCAGCAATGGAGATTCCAAGAAATCAGGAATTGGAGCTGGTGCTATAGCAGGTATAATCATTTCATTACTAGTAGTTACAGCTCTTGTGGCTTTCTTCTTAGTCAAAAGAAGAAGAAGATCAAAGAGATCATCATCTATGGACATTGAGAAAACTGACAACCAGCCTTTCACTCTTCCTCCAAGCGACTTTCACGAAAACAATTCTATTCAGAGTTCTTCATCAATTGAGACAAAGAAACTTGATACTTCCTTGTCTATTAATCTCCGTCCTCCACCAGCTGATCGATCATTTGATGATGATGAGGATTCTACGAGAAAGCCTATAGTTGTCAAGAAATCCACCGTGGCTGTTCCCTCGAATGTGAGAGTTTACTCAGTTGCTGATCTTCAGATTGCCACTGCCAGTTTCAGTGTTGATAATCTTCTTGGAGAAGGCACTTTTGGAAGAGTATACAGAGCTGAGTTTAACAATGGAAAGGTTCTTGCTGTGAAGAAGATTGATTCATCTGCTCTTCCACATAGCATGACTGATGATTTCACCGAAATAGTATCGAAAATAGCCGTTTTGGATCATCCAAATGTGACAAAGCTTGTTGGCTACTGTGCTGAACACGGACAACATCTCCTGGTCTATGAGTTCCACAGCAAAGGATCGTTACATGACTTCCTACACTTATCAGAAGAAGAAAGCAAAGCATTGGTGTGGAACTCGCGAGTCAAGGTCGCACTTGGGACTGCACGGGCAATAGAGTACTTGCATGAAGTTTGTTCACCGTCTATAGTTGACAAGAACATCAAATCAGCCAATATTTTGCTTGATTCGGAGATGAATCCTCACTTATCAGACACAGGTCTCGCAAGCTTCCTCCCCACAGCAAATGAGTTACTAAACCAAACCGATGAAGGTTATAGCGCACCGGAAGTATCAATGTCAGGTCAATACTCTTTGAAGAGTGATGTTTACAGTTTTGGAGTAGTGATGCTTGAACTTTTAACCGGGAGGAAACCATTCGACAGCACAAGGTCAAGATCTGAGCAGTCATTGGTTAGATGGGCGACACCACAGCTTCATGACATTGATGCTTTAGGCAAAATGGTTGATCCAGCTCTTGAAGGACTTTATCCGGTTAAATCTCTTTCTCGGTTTGCAGATGTTATTGCTCTCTGCGTCCAGCCAGAGCCAGAGTTTAGACCACCAATGTCTGAAGTTGTGCAGTCACTGGTTGTGTTAGTGCAGAGAGCTAACATGAGCAAGAGAACTGTTGGAGTTGATCCATCACAGCGTTCTGGTAGTGCTGAGCCAAGCAACGATTACATGTAAACCCATTACCACAGAGAGAGAAAAAAAGAACACTTTGCTCCCTATGGGATGAAGTCATTGTTTTTATTGTAATATGTTTGATAAACCTTCACACAGTATATTATCCCCATTGTATTTTGTTGTAATGTGTTTCCAAATTTGTAGCTTTTAGATCATTGAAATGAACAAATATTCTTTCTTGTGTAAAAAAAAAAAAAAAAAA Deduced amino acidsequence of BnPK-4 from Brassica napus (SEQ ID NO:20)MMENRVVVVAALFAVCIVGFEFSFIHGATDASDTSALNMLFTSMHSPGQLTQWTASGGDPCVQNWRGVTCSKSRITQLKLSGLELSGTLGYMLDKLTSLTELDLSSNNLGGDLPYQLPPNLQRLNLANNQFTGAAQYSISNMASLKYLNLGHNQFKGQVAVDFSKLTSLTTLDFSFNSFTSSLPGTFTSLTSLKSLYLQNNQFSGTLNVLAGLPLETLNIANNDFTGWIPSTLKGTNLIKDGNSFNNGPAPPPPPGTPPIHRSPSHKSGGGSNRDSTSNGDSKKSGIGAGAIAGIIISLLVVTALVAFFLVKRRRRSKRSSSMDIEKTDNQPFTLPPSDFHENNSIQSSSSIETKKLDTSLSINLRPPPADRSFDDDEDSTRKPIVVKKSTVAVPSNVRVYSVADLQIATASFSVDNLLGEGTFGRVYRAEFNNGKVLAVKKIDSSALPHSMTDDFTEIVSKIAVLDHPNVTKLVGYCAEHGQHLLVYEFHSKGSLHDFLHLSEEESKALVWNSRVKVALGTARAIEYLHEVCSPSIVDKNIKSANILLDSEMNPHLSDTGLASFLPTANELLNQTDEGYSAPEVSMSGQYSLKSDVYSFGVVMLELLTGRKPFDSTRSRSEQSLVRWATPQLHDIDALGKMVDPALEGLYPVKSLSRFADVIALCVQPEPEFRPPMSEVVQSLVVLVQRANMSKRTVGV DPSQRSGSAEPSNDYM*Nucleotide sequence of GmPK-1 from Glycine max (SEQ ID NO:21)TTTAGAGAGAGAAAGAGTGTGAGTGTTGTGTTGAGTGCAGTTTCTTTCTCACATGGCCTCTATGCCGTTGGGGCCGCAGCAACAGCTTCCACCGCCGCCGCCGCAACAACCGCCGCCAGCGGAGAATGACGCGATGAAAGTGGACTCTCGCGGCGGCTCCGACGCCGGCACCGAAAAGGAAATGTCAGCTCCTGTCGCAGATGGTAATGATGCACTCACTGGTCACATAATCTCAACCACAATTGCAGGCAAAAATGGCGAACCTAAACAAACCATCAGTTACATGGCCGAACGTGTTGTTGGCACTGGATCATTTGGCATTGTTTTCCAGGCGAAGTGCTTGGAGACTGGCGAGGCAGTGGCTATAAAGAAGGTCTTGCAGGACAGGCGATACAAAAATCGTGAACTGCAGTTAATGCGCGTGATGGATCACCCAAATATAATTTCCTTGAGTAACTATTTCTTCTCTACAACAAGTAGAGATGAACTTTTTCTGAACTTGGTGATGGAATATGTCCCTGAGACGATCTTCCGTGTTATAAAGCACTACAGTAGCATGAAACAGAGAATGCCCCTAATCTATGTGAAATTATATACATATCAAATCTTTAGGGGACTGGCGTATATCCATACTGTACCAGGAATCTGCCATAGGGATTTGAAGCCTCAAAATCTTTTGGTTGATCGACTCACACACCAAGTCAAGCTCTGTGATTTTGGGAGTGCAAAAGTTCTGGTGGAGGGTGAATCAAACATTTCATACATATGTTCACGGTACTATCGTGCCCCAGAGCTAATATTTGGTGCGGCAGAATACACAACTTCTGTTGATATTTGGTCCGCTGGTTGTGTCCTTGCGGAACTTCTTCTAGGCCAGCCTTTGTTCCCAGGAGAAAATCAGGTTGACCAACTCGTGGAAATTATCAAGATTCTTGGCACTCCTACTCGAGAAGAAATTCGATGCATGAATCCTAATTATACAGATTTCAGATTCCCCCATATCAAAGCTCATCCTTGGCATAAGGTTTTTCACAAGCGAATGCCTCCTGAAGCAATTGACCTTGCATCAAGGCTTCTCCAATATTCCCCAAAACTTCGTTACAGTGCAGTGGAAGCAATGGCACATCCTTTCTTTGACGAGCTTCGCGAGCCCAATGCCCGGCTACCTAATGGTCGTCCACTGCCTCCACTTTTCAACTTTAAACAGGAATTAGATGGAGCGCCCCCTGAACTGCTTCCTAAGCTCATCCCAGAGCATGTCAGGCGGCAAACCCAAATGTAAAGAGATAGTAAAACATAGAGTGAACTGTTCTAGTGGATTAGTGTGAAATACATGAGAGCTTGCTTGTGGTCAATAGAACAGGGGTTAGGCCCAAATATGCAGTTTTTCTCCCCCTTGTGAAGATGTATACATGTGCTGGAAAACTCAGTGTAACCCGGAAATGTAGATTATGTCTAATGTCTAATATTTCATTCTAGTTAAAAAAAAAAAAAAAAAA Deduced amino acidsequence of GmPK-1 from Glycine max (SEQ ID NO:22)MASMPLGPQQQLPPPPPQQPPPAENDAMKVDSRGGSDAGTEKEMSAPVADGNDALTGHIISTTIAGKNGEPKQTISYMAERVVGTGSFGIVFQAKCLETGEAVAIKKVLQDRRYKNRELQLMRVMDHPNIISLSNYFFSTTSRDELFLNLVMEYVPETIFRVIKHYSSMKQRMPLIYVKLYTYQIFRGLAYIHTVPGICHRDLKPQNLLVDRLTHQVKLCDFGSAKVLVEGESNISYICSRYYRAPELIFGAAEYTTSVDIWSAGCVLAELLLGQPLFPGENQVDQLVEIIKILGTPTREEIRCMNPNYTDFRFPHIKAHPWHKVFHKRMPPEAIDLASRLLQYSPKLRYSAVEAMAHPFFDELREPNARLPNGRPLPPLFNFKQELDGAPPELLPKLIP EHVRRQTQM* Nucleotidesequence of GmPK-2 from Glycine max (SEQ ID NO:23)AGACACCACAAAGTGTAACTTGAGTGATTATATCTGATGAGTGCAGAAAGAAGGGAGGATTGTTGGTGATCGATCATCGATCATCGATCATCGATCATCGATGGCGTCTGCTAGCCTTGGAAGTGGTGGGGTGGGCAGTTCCAGGTCTGTTAATGGTGGCTTCAGGGGTTCTTCCAGTTCCGTCGATTGGCTTGGCAGAGAGATGCTTGAGATGTCTTTGAGAGACCACGAGGACGATAGAGATAGTGAGCCTGACATCATTGATGGTTTGGGTGCTGAGACTGGTCACGTGATAAGAACCAGCGTTGGTGGCCGAAATGGTCAATCTAAGCAGAATGTTAGTTATATTTCTGAGCATGTTGTGGGAACAGGCTCTTTTGGTGTTGTTTTTCAAGCCAAATGTAGAGAAACGGGAGAAATTGTGGCCATCAAGAAAGTTCTCCAGGACAAGCGCTACAAGAATAGAGAGTTACAAATTATGCAAATGCTGGATCATCCAAATATTGTTGCCCTTAGGCATTGTTTCTATTCAACGACTGACAAAGAAGAAGTTTACTTGAATCTTGTACTTGAATATGTTCCTGAAACTGTGAATCGCATCGCCAGGAGCTATAGCAGGATTAACCAGCGAATGCCTTTAATATATGTAAAGCTTTATACCTACCAGATTTGCAGGGCCCTTGCTTATATACATAACTGCATTGGTATATGTCATCGTGACATCAAACCTCAGAACCTACTTGTGAACCCGCACACTCATCAGCTGAAACTATGTGATTTTGGGAGTGCAAAAGTGTTGGTGAAAGGAGAACCTAATGTTTCTTACATCTGTTCAAGATACTACCGTGCTCCGGAACTTATATTTGGGGCCACTGAATATACAACTGCCATAGATATATGGTCAACTGGTTGTGTAATGGCTGAATTACTTCTTGGACAGCCCTTGTTTCCTGGAGAGAGTGGAGTTGATCAGCTAGTTGAAATCATCAAGGTTTTGGGAACTCCAACCAGGGAGGAGATAAAGTGCATGAACCCAAATTATACTGAATTTAAGTTTCCACAGATAAAACCTCATCCATGGCACAAGGTTTTTCAGAAACGTTTACCCCCAGAAGCAGTGGACCTTGTCTGTAGGTTCTTTCAGTACTCTCCCAATTTGAGATGCACTGCATTGGAAGCTTGCATTCATCCATTTTTTGATGAATTGAGGGACCCAAACACCCGCCTTCCTAATGGTCGACCACTTCCTCCACTGTTTAATTTTAAACCTCAGGAACTTTCTGGTGTACCCCCTGATGTCATCAATCGGCTTATTCCAGAGCATGCGCGTAAACAGAACTTATTTATGGCTTTGCACACCTAGCAATTCCCGTACCCTCCTAAGTTGTCGTCACTTACTAGCAGGTTGTAAATTATCCGGTTTATCCGAGAAAAACTCCACAGAAAGAGTTACTAGGATTATATTATTATTATATAATATGAAAAGTTTCTTTTTTCTTTTTTGGAAAAAAAAAAAAAAAAAA Deduced amino acid sequence of GmPK-2 fromGlycine max (SEQ ID NO:24)MASASLGSGGVGSSRSVNGGFRGSSSSVDWLGREMLEMSLRDHEDDRDSEPDIIDGLGAETGHVIRTSVGGRNGQSKQNVSYISEHVVGTGSFGVVFQAKCRETGEIVAIKKVLQDKRYKNRELQIMQMLDHPNIVALRHCFYSTTDKEEVYLNLVLEYVPETVNRIARSYSRINQRMPLIYVKLYTYQICRALAYIHNCIGICHRDIKPQNLLVNPHTHQLKLCDFGSAKVLVKGEPNVSYICSRYYRAPELIFGATEYTTAIDIWSTGCVMAELLLGQPLFPGESGVDQLVEIIKVLGTPTREEIKCMNPNYTEFKFPQIKPHPWHKVFQKRLPPEAVDLVCRFFQYSPNLRCTALEACIHPFFDELRDPNTRLPNGRPLPPLFNFKPQELSGVPPDV INRLIPEHARKQNLFMALHT*Nucleotide sequence of GmPK-3 from Glycine max (SEQ ID NO:25)AGAGAGAGAAACGAAGAAGAAGAGTGTTTCTCACATCACATGGCCTCCTTGCCCTTGGGGCACCACCACCACCACCACAAACCGGCGGCGGCGGCTATACATCCGTCGCAACCGCCGCAGTCTCAGCCGCAACCCGAAGTTCCTCGCCGGAGCTCCGATGTGGAGACCGATAAGGATATGTCAGCTACTGTCATTGAGGGGAATGATGCTGTCACTGGCCACATAATCTCCACCACAATTGGAGGCAAAAATGGGGAACCTAAAGAGACCATCAGTTACATGGCAGAACGTGTTGTTGGCACTGGATCATTTGGAGTTGTTTTTCAGGCAAAGTGCTTGGAGACTGGAGAAGCAGTGGCTATTAAAAAGGTCTTGCAAGACAGGCGGTACAAAAATCGTGAATTGCAGTTAATGCGCTTAATGGATCACCCTAATGTAATTTCCCTGAAGCACTGTTTCTTCTCCACAACAAGCAGAGATGAACTTTTTCTAAACTTGGTAATGGAATATGTTCCCGAATCAATGTACCGAGTTATAAAGCACTACACTACTATGAACCAGAGAATGCCTCTCATCTATGTGAAACTGTATACATATCAAATCTTTAGGGGATTAGCATATATCCATACCGCACTGGGAGTTTGCCATAGGGATGTGAAGCCTCAAAATCTTTTGGTTCATCCTCTTACTCACCAAGTTAAGCTATGTGATTTTGGGAGTGCCAAAGTTCTGGTCAAGGGTGAATCAAACATTTCATACATATGTTCACGTTACTATCGGGCTCCAGAACTAATATTTGGTGCAACAGAATACACAGCTTCTATTGATATCTGGTCAGCTGGTTGTGTTCTTGCTGAACTTCTTCTAGGACAGCCATTATTTCCTGGAGAAAACCAAGTGGACCAACTTGTGGAAATTATCAAGGTTCTTGGTACTCCAACACGCGAGGAAATCCGTTGTATGAACCCAAATTATACAGAGTTTAGATTCCCTCAGATTAAAGCTCATCCTTGGCACAAGGTTTTCCACAAGCGAATGCCTCCTGAAGCAATTGACCTTGCATCAAGGCTTCTCCAATATTCACCTAGTCTCCGCTGCACTGCGCTGGAAGCATGTGCACATCCTTTCTTTGATGAGCTTCGCGAACCAAATGCCCGGCTACCTAATGGCCGTCCACTGCCCCCACTTTTCAACTTCAAACAGGAGTTAGCTGGAGCATCACCTGAACTGATCAATAGGCTCATCCCAGAGCATATTAGGCGGCAGATGGGTCTCAGCTTCCCGCATTCTGCCGGTACATAGATGTAAAGGGATAATGAAACGATGAGTCAACCTACATAGTGATCGATGTGAATCAACAGAAGGGCTGTTTGAGGCCTATGTATAACTGGGAGTCCCAACATAATATGCAGTTTTTCCTCCCCCTTGTGAAGATGTATACATGTGTTGGTTGCTCGGTAAAGCTTGAAAGTTGGTGATTCTGTGTAGTATTTCATTCAAGTTAAAGCATACTTATCCCTGCATCTGTATATTGTTTTGGTCAGATTTCAGAAAGCTAGGAGTATAAAATGATAGCAATCATGTCTTCATAGGTAGAGGGGCCCAGCTGAATTGAGGGGCCCCTATAGTAGTTTGGCTTGCTTTTTATGAGATTAAATTCAGGATGTCGTTTATATTATGTTTATAACAATCTCTTGATTCAAAACAAGAAATTTTCTCGTTGTTGAAAAAAAAAAAAAAAAAA Deduced amino acid sequenceof GmPK-3 from Glycine max (SEQ ID NO:26)MASLPLGHHHHHHKPAAAAIHPSQPPQSQPQPEVPRRSSDVETDKDMSATVIEGNDAVTGHIISTTIGGKNGEPKETISYMAERVVGTGSFGVVFQAKCLETGEAVAIKKVLQDRRYKNRELQLMRLMDHPNVISLKHCFFSTTSRDELFLNLVMEYVPESMYRVIKHYTTMNQRMPLIYVKLYTYQIFRGLAYIHTALGVCHRDVKPQNLLVHPLTHQVKLCDFGSAKVLVKGESNISYICSRYYRAPELIFGATEYTASIDIWSAGCVLAELLLGQPLFPGENQVDQLVEIIKVLGTPTREEIRCMNPNYTEFRFPQIKAHPWHKVFHKRMPPEAIDLASRLLQYSPSLRCTALEACAHPFFDELREPNARLPNGRPLPPLFNFKQELAGASPELINR LIPEHIRRQMGLSFPHSAGT*Nucleotide sequence of GmPK-4 from Glycine max (SEQ ID NO:27)GAGTTTCAAAGGTTGTTGGTGTGCATCACCACCTGCATTCTATGTTGGATGCCCAATGGTGCCACTGCCGCCACAGATCCAAATGATGCTGCTGCTGTGAGATTTTTGTTTCAAAATATGAACTCACCACCCCAGCTAGGTTGGCCTCCTAATGGTGATGATCCATGTGGACAATCTTGGAAAGGCATTACTTGCTCTGGCAATCGTGTTACAGAGATTAAGTTATCTAATCTTGGACTAACTGGATCGTTGCCTTATGGATTACAAGTCTTGACATCTTTGACCTACGTAGACATGAGTAGCAACAGTCTTGGTGGCAGCATACCGTACCAACTTCCTCCATATTTGCAGCACTTAAATCTTGCTTATAACAACATCACAGGGACAGTACCTTATTCGATTTCTAACTTGACTGCTCTTACTGACCTGAATTTTAGTCACAATCAGCTCCAGCAAGGACTGGGTGTTGACTTTCTTAATCTTTCTACTCTCTCCACATTGGATCTCTCTTTCAATTTTCTAACAGGTGACCTCCCTCAGACTATGAGCTCACTTTCACGCATAACCACCATGTATCTGCAAAATAACCAGTTTACAGGCACTATTGATGTCCTTGCTAATCTGCCTCTGGATAATCTGAATGTGGAAAATAATAATTTTACTGGATGGATACCAGAACAGTTGAAGAACATAAACCTACAGACCGGTGGTAATGCATGGAGCTCAGGGCCTGCACCCCCACCTCCTCCTGGGACACCTCCAGCACCTAAAAGCAACCAGCACCACAAGTCTGGTGGTGGAAGCACCACCCCCTCAGATACTGCCACTGGCAGCAGCTCAATTGACGAGGGAAAAAAATCTGGTACAGGAGGTGGTGCCATAGCCGGAATTGTGATCTCTGTCATAGTTGTGGGGGCAATAGTAGCATTCTTTCTGGTGAAGAGAAAATCCAAGAAGTCATCTTCTGATTTAGAAAAGCAGGATAATCAGTCCTTTGCTCCACTTCTTTCAAATGAAGTGCATGAAGAAAAGTCCATGCAAACTTCCTCTGTAACAGACTTGAAGACGTTTGATACTTCTGCCTCAATAAATCTTAAACCCCCACCTATTGACCGTCATAAATCATTTGATGATGAAGAATTCTCCAAGAGGCCCACAATTGTGAAGAAGACTGTAACAGCTCCTGCAAATGTGAAATCATATTCTATTGCTGAACTGCAGATTGCTACTGGCAGCTTCAGTGTGGATCACCTTGTTGGCGAGGGATCTTTTGGGCGTGTTTACCGTGCTCAATTTGATGATGGACAGGTTCTTGCAGTGAAGAAGATAGATTCATCTATCCTTCCCAATGATTTGACAGATGATTTTATACAAATAATTTCAAACATCTCCAATTTACATCATCCAAATGTGACAGAGCTTGTAGGTTATTGCTCAGAGTATGGACAACACCTCTTGGTCTATGAGTTTCATAAAAATGGATCACTGCATGACTTCCTTCACCTATCAGATGAATATAGTAAACCATTGATATGGAATTCCCGTGTCAAGATTGCTTTGGGGACTGCACGTGCTCTAGAGTACCTACATGAAGTTAGTTCGCCATCAGTTGTTCATAAGAATATTAAGTCAGCCAACATATTACTTGATACAGAACTTAATCCTCATCTTTCAGATAGTGGATTGGCAAGTTATATTCCAAATGCCGACCAGATATTGAATCATAATGTTGGATCTGGATATGATGCACCTGAAGTTGCCTTGTCTGGTCAGTATACTTTGAAAAGTGATGTCTACAGCTTTGGAGTCGTCATGTTGGAACTTCTCAGTGGACGGAACCCTTTTGATAGCTCAAGGCCAAGATCTGAGCAGTCTTTGGTTCGATGGGCAACACCTCAACTCCATGATATTGATGCATTGGCTAAAATGGTTGATCCTGCAATGAAAGGGTTATATCCTGTTAAGTCTCTTTCTCGATTTGCCGATGTTATTGCTCTTTGCGTTCAGCCGGAGCCAGAATTCCGACCACCGATGTCAGAAGTGGTTCAAGCACTGGTGCGACTAGTGCAGCGAGCTAACATGAGCAAGCGAACATTTAGTAGTAGTGATCATGGAGGATCCCAACGAGGGAGTGATGAGCCAGTTCTACGAGACATCTAAATCCCAAAGCAAATGTAGTTATATTTTTCTCCCAAGCTAGTTCGGTTATTTGTAATATAATTTCCAATAGTTGCAAATTTGAATTGATGGGTTCCATATTCTGTTGATACCTATGTAAACCTGTCCAAATCAGCTTATTACAATGACAGTAACGGTTGCACTGGCAAAAAAAAAAAAAAAAA Deduced amino acid sequenceof GmPK-4 from Glycine max (SEQ ID NO:28)MPNGATAATDPNDAAAVRFLFQNMNSPPQLGWPPNGDDPCGQSWKGITCSGNRVTEIKLSNLGLTGSLPYGLQVLTSLTYVDMSSNSLGGSIPYQLPPYLQHLNLAYNNITGTVPYSISNLTALTDLNFSHNQLQQGLGVDFLNLSTLSTLDLSFNFLTGDLPQTMSSLSRITTMYLQNNQFTGTIDVLANLPLDNLNVENNNFTGWIPEQLKNINLQTGGNAWSSGPAPPPPPGTPPAPKSNQHHKSGGGSTTPSDTATGSSSIDEGKKSGTGGGAIAGIVISVIVVGAIVAFFLVKRKSKKSSSDLEKQDNQSFAPLLSNEVHEEKSMQTSSVTDLKTFDTSASINLKPPPIDRHKSFDDEEFSKRPTIVKKTVTAPANVKSYSIAELQIATGSFSVDHLVGEGSFGRVYRAQFDDGQVLAVKKIDSSILPNDLTDDFIQIISNISNLHHPNVTELVGYCSEYGQHLLVYEFHKNGSLHDFLHLSDEYSKPLIWNSRVKIALGTARALEYLHEVSSPSVVHKNIKSANILLDTELNPHLSDSGLASYIPNADQILNHNVGSGYDAPEVALSGQYTLKSDVYSFGVVMLELLSGRNPFDSSRPRSEQSLVRWATPQLHDIDALAKMVDPAMKGLYPVKSLSRFADVIALCVQPEPEFRPPMSEVVQALVRLVQRANMSKRTFSSSDHGGSQRGSDEPVL RDI* Nucleotidesequence of OsPK-1 from Oryza sativa (SEQ ID NO:29)ACCACACAAAAAAGCAAAACAGAGAGAACAACTGTTACTCACACACGCCATGGGTAAATGAATGGTTTTTGAGCAACAGCAGTTAAAAGAGAAAAGGGATTCAGCGAAGATGACATCGGTTGGTGTGGCACCAACTTCGGGTTTGAGAGAAGCCAGTGGGCATGGAGCAGCAGCTGCGGATAGATTGCCAGAGGAGATGAACGATATGAAAATTAGGGATGATAGAGAAATGGAAGCCACAGTTGTTGATGGCAACGGAACGGAGACAGGACATATCATTGTGACTACCATTGGGGGTAGAAATGGTCAGCCCAAGCAGACTATAAGCTACATGGCAGAGCGTGTTGTAGGGCATGGATCATTTGGAGTTGTCTTCCAGGCTAAGTGCTTGGAAACCGGTGAAACTGTGGCTATCAAAAAGGTTCTTCAAGATAAGAGGTACAAGAACCGGGAGCTGCAAACAATGCGCCTTCTTGACCACCCAAATGTTGTCGCTTTGAAGCACTGTTTCTTTTCAACCACTGAAAAGGATGAACTATACCTCAATTTGGTACTTGAATATGTTCCTGAAACAGTTAATCGTGTGATCAAACATTACAACAAGTTAAACCAAAGGATGCCGCTGATATATGTGAAACTCTATACATACCAGATCTTTAGGGCGTTATCTTATATTCATCGTTGTATTGGAGTCTGCCATCGGGATATCAAGCCTCAAAATCTATTGGTCAATCCACACACTCACCAGGTTAAATTATGTGACTTTGGAAGTGCAAAGGTTTTGGTAAAAGGCGAACCAAATATATCATACATATGTTCTAGATACTATAGAGCACCTGAGCTCATATTTGGCGCAACTGAATATACTTCAGCCATTGACATCTGGTCTGTTGGATGTGTTTTAGCTGAGCTGCTGCTTGGACAGCCTCTGTTCCCTGGTGAGAGTGGAGTTGATCAACTTGTTGAGATCATCAAGGTTCTGGGCACTCCAACAAGGGAAGAGATTAAGTGCATGAACCCTAATTATACAGAATTTAAATTCCCACAGATTAAAGCACATCCATGGCACAAGATCTTCCATAAGCGCATGCCTCCAGAGGCTGTTGATTTGGTATCAAGACTACTACAATACTCCCCTAACTTGCGGTGCACAGCTTTTGATGCCTTGACGCATCCTTTCTTCGACGAGCTTCGTGATCCAAATACTCGCTTGCCAAATGGCCGATTCCTTCCACCACTATTTAATTTCAAATCCCATGAACTGAAAGGAGTCCCATCTGAGATTTTGGTGAAATTGGTTCCAGAGCATGCAAGGAAGCAATGCCCGTTTCTAGGCTCGTGAAGTGTTGTTTCCATATGAGAATGCTGCGCTTTCCTTTTCTATTTAATATGATATTTTTGTTGGTATCTTTATTGTATTCGGTTGCCCTGTAAAAGCAGATTTAGAGATACATGCTACTCATTATCACCCAACCCCCGATGGTTATGTAGAATACCCTGTTTCCTGTATCACAGCAGATTGTAACATACAATAGAGGACAAAATGTCTGCAATTATCTAAATGTTGCATCAATATTTGTATTTGTTGAGGCAAAAAAAA AAAAAAAAAA Deducedamino acid sequence of OsPK-1 from Oryza sativa (SEQ ID NO:30)MVFEQQQLKEKRDSAKMTSVGVAPTSGLREASGHGAAAADRLPEEMNDMKIRDDREMEATVVDGNGTETGHIIVTTIGGRNGQPKQTISYMAERVVGHGSFGVVFQAKCLETGETVAIKKVLQDKRYKNRELQTMRLLDHPNVVALKHCFFSTTEKDELYLNLVLEYVPETVNRVIKHYNKLNQRMPLIYVKLYTYQIFRALSYIHRCIGVCHRDIKPQNLLVNPHTHQVKLCDFGSAKVLVKGEPNISYICSRYYRAPELIFGATEYTSAIDIWSVGCVLAELLLGQPLFPGESGVDQLVEIIKVLGTPTREEIKCMNPNYTEFKFPQIKAHPWHKIFHKRMPPEAVDLVSRLLQYSPNLRCTAFDALTHPFFDELRDPNTRLPNGRFLPPLFNFKSHELKGVPSEILVKLVPEHARKQCPFLGS*

1. A transgenic plant cell transformed with an isolated polynucleotideselected from the group consisting of: i. a polynucleotide having asequence as set forth in SEQ ID NO:2; and ii. a polynucleotide encodinga polypeptide having a sequence as set forth in SEQ ID NO:3.
 2. Theplant cell of claim 1, wherein the polynucleotide has the sequence asset forth in SEQ ID NO:2.
 3. The plant cell of claim 1, wherein thepolynucleotide encodes the polypeptide having the sequence as set forthin SEQ ID NO:3.
 4. A transgenic plant transformed with an isolatedpolynucleotide selected from the group consisting of: a) apolynucleotide having a sequence as set forth in SEQ ID NO:2; and b) apolynucleotide encoding a polypeptide having a sequence as set forth inSEQ ID NO:3.
 5. The plant of claim 4, wherein the polynucleotide has thesequence as set forth in SEQ ID NO:2.
 6. The plant of claim 4, whereinpolynucleotide encodes the polypeptide having the sequence as set forthin SEQ ID NO:3.
 7. The plant of claim 4, wherein the plant is a monocot.8. The plant of claim 4, wherein the plant is a dicot.
 9. The plant ofclaim 4, wherein the plant is selected from the group consisting ofmaize, wheat, rye, oat, triticale, rice, barley, soybean, peanut,cotton, rapeseed, canola, manihot, pepper, sunflower, tagetes, potato,tobacco, eggplant, tomato, Vicia species, pea, alfalfa, coffee, cacao,tea, Salix species, oil palm, coconut, perennial grasses, and a foragecrop plant.
 10. A seed which is true breeding for a transgene comprisinga polynucleotide selected from the group consisting of: a) apolynucleotide having a sequence as set forth in SEQ ID NO:2; and b) apolynucleotide encoding a polypeptide having a sequence as set forth inSEQ ID NO:3.
 11. The seed of claim 10, wherein the polynucleotide hasthe sequence as set forth in SEQ ID NO:2.
 12. The seed of claim 10,wherein the polynucleotide encodes the polypeptide having the sequenceas set forth in SEQ ID NO:3.
 13. An isolated nucleic acid comprising apolynucleotide selected from the group consisting of: a) apolynucleotide having a sequence as set forth in SEQ ID NO:2; and b) apolynucleotide encoding a polypeptide having a sequence as set forth inSEQ ID NO:3.
 14. The isolated nucleic acid of claim 13, wherein thepolynucleotide has the sequence as set forth in SEQ ID NO:2.
 15. Theisolated nucleic acid of claim 13, wherein the polynucleotide encodesthe polypeptide having the sequence as set forth in SEQ ID NO:3.
 16. Amethod of producing a drought-tolerant transgenic plant, the methodcomprising the steps of: a) transforming a plant cell with an expressionvector comprising a polynucleotide encoding a polypeptide having asequence as set forth in SEQ ID NO:3; b) growing the transformed plantcell to generate transgenic plants; and c) screening the transgenicplants generated in step b) to identify a transgenic plant thatexpresses the polypeptide and exhibits increased tolerance to droughtstress as compared to a wild type variety of the plant.
 17. The methodof claim 16, wherein the polynucleotide has a sequence as set forth inSEQ ID NO:2.